link
stringlengths 41
45
| date
stringlengths 9
9
| paper
dict | reviews
listlengths 1
6
| version
int64 1
5
| main
stringlengths 38
42
|
|---|---|---|---|---|---|
https://f1000research.com/articles/1-8/v1
|
02 Aug 12
|
{
"type": "Commentary",
"title": "Innate B cells: oxymoron or validated concept?",
"authors": [
"Carl F Ware",
"Chris Benedict",
"Chris Benedict"
],
"abstract": "B lymphocytes promote the initial innate interferon response to viral pathogens without the need for antigen receptor activation. B cell dependent IFN production requires the cytokine, lymphotoxin-β. The LTβ pathway is well known to regulate lymphoid organogenesis and homeostasis by differentiating stromal cells and macrophages. However, in response to viral pathogens these same B cell-regulated populations rapidly produce type 1 interferons. Thus, B cells act as innate effector cells via LTβ homeostatic pathways, which serve as innate host barriers to viral pathogens.",
"keywords": [
"The B cell is an icon of the adaptive immune system",
"secreting a specific antibody that prevents re-infection by pathogens. Although some B cell subsets (e.g.",
"B1 cells) show characteristics of \"innate\" cells (defined here as cells that do not utilize antibody or antigen receptor genes)",
"the concept of an \"innate B cell\" somehow doesn't register. However",
"accumulating evidence validates another view of B cells",
"one as an innate effector cell initiating the earliest response against viral pathogens",
"independently of antibody. In two studies",
"B cells were shown to control the initial type 1 IFN response to very different viral pathogens in lymphoid tissues",
"cytomegalovirus (CMV",
"a β-herpesvirus with a large DNA based genome)1 and vesicular stomatitis virus (VSV",
"a small RNA virus that causes lympho-neurotropic pathogenicity)2."
],
"content": "Lymphotoxin-β pathway and innate B cells\n\nThe B cell is an icon of the adaptive immune system, secreting a specific antibody that prevents re-infection by pathogens. Although some B cell subsets (e.g., B1 cells) show characteristics of \"innate\" cells (defined here as cells that do not utilize antibody or antigen receptor genes), the concept of an \"innate B cell\" somehow doesn't register. However, accumulating evidence validates another view of B cells, one as an innate effector cell initiating the earliest response against viral pathogens, independently of antibody. In two studies, B cells were shown to control the initial type 1 IFN response to very different viral pathogens in lymphoid tissues, cytomegalovirus (CMV, a β-herpesvirus with a large DNA based genome)1 and vesicular stomatitis virus (VSV, a small RNA virus that causes lympho-neurotropic pathogenicity)2.\n\nSchneider et al.1 established the B cell dependence of the IFNβ response to infection with CMV. This innate IFN defense mechanism was surprisingly independent of Toll-like receptor pathways, but required the Lymphotoxin (LT)-β receptor signaling pathway, part of the larger superfamily of cytokines related to TNF3,4. Conditional deletion of the LTβ gene in B cells, but not T cells, provided the key evidence pinpointing the involvement of LTβ in B cells in the initial IFN response to CMV. The LT-IFN response occurs rapidly, initiating within a couple of hours after infection, well before adaptive immunity could contribute. Expression of the IFNβ gene occurred primarily in virus-infected stromal cells in the spleen and accounted for the majority of the circulating IFNαβ. Blocking the LT-IFN pathway resulted in destruction of the splenic architecture and an apoptotic collapse of T and B lymphocytes5.\n\nMoseman et al.2 demonstrated the critical role of the B cell dependent LT-IFN defense pathway in response to VSV. Importantly, antibody deficient μMT and DHLMP2a mice revealed IFNβ expression in response to VSV occurred independently of B cell antigen receptor. In the absence of LTβ or IFN signaling, VSV infected the lymphatic neurons and spread into the central nervous system with ensuing paralysis. These results provide strong evidence for the innate action of B cells through the LT-IFN pathway. The effectiveness of the LT-IFN pathway against two distinct pathogens suggests a more generalized role in host defense.\n\n\nThe architecture of host defense\n\nThe current evidence indicates the innate B cell driven LT-IFN pathway operates during infections in lymphoid tissues. A convergence of recent results may explain this observation. Mouse CMV productively infects reticular fibroblasts in the splenic marginal zone, but also subcapsular sinus macrophages in lymph nodes that express high CD169+ (SIGLEC1) (Figure 1)6. Interestingly, CD169+ macrophages uniquely support VSV infection in lymph nodes and provide the primary source of IFNα during the initial phase of infection with VSV7. Importantly, these CD169+ macrophages require the LTαβ-LTβR pathway to populate the subcapsular regions in lymph nodes and the marginal sinus of the spleen8,9. LTβ receptor signaling regulates stromal cell expression of homeostatic chemokines (e.g., CXCL13, CCL21) that help to position CD169+ macrophages in lymph nodes and spleen. In the absence of LTβR signaling, CD169+ subcapsular macrophages no longer reside in lymph nodes, depriving the virus of a permissive cell for replication, with a commensurate loss in IFNα production. The mechanism underlying the permissiveness of the CD169+ macrophages is not entirely understood, however Khanna and Lefrancois10 point out that these macrophages have limited capacity to respond to IFN due to expression of Usp1811 encoding an ISG15-deconjugating peptidase that destabilizes multiple antiviral proteins induced by IFN12.\n\nB cells express the TNF-related ligand LTαβ that specifically engages the LTβR expressed in lymphoid tissue stromal cells (pink) and myeloid lineage cells including subcapsular macrophages (green). The B cell to stromal cell interaction maintains the homeostasis of lymphoid tissues through secretion of chemokines and IL7, which enhance the expression of LTαβ. Cytomegalovirus (CMV) infects stromal cells (ERTR7+ fibrocytes) in the splenic marginal zone from which IFNβ is rapidly expressed and secreted. B cell expression of LTαβ is also required for CD169+ subcapsular macrophages in lymph nodes. Vesicular stomatitis virus (VSV) infects subcapsular macrophages inducing production of IFNα. Virus replication and progeny are produced (red arrows) in the permissive stromal cells or CD169+ macrophages. IFNαβ protect uninfected cells in the surrounding microenvironment.\n\nLymphoid organs provide the structural environment that positions key cells, such as the sinus lining macrophages, directly in the flow of lymph and blood in order to capture pathogens13. Yet, intentionally providing a pathogen with a source of permissive cells seems counterintuitive as a defense strategy. However, amplifying the level of viral antigens to increase presentation to adaptive immune cells could counterbalance this potential danger. Moreover, the powerful selective pressure that the IFN system places on the pathogen is relieved in the permissive macrophage, potentially limiting the emergence of mutant viruses resistant to IFN. Neighboring cells that remain responsive to IFN signaling should be protected, corralling the pathogen within this macrophage-populated niche.\n\nThe Lymphotoxin-β pathway orchestrates the embryonic development of lymph nodes and Peyer’s Patches14–16. In the adult, B lymphocytes constitutively expressing LTβ are the primary cells responsible for the maintenance of the microarchitecture of the spleen and lymph nodes. Thus, this LTβ-dependent developmental pathway is reflected in the adult as an innate B cell host defense mechanism. Recent evidence indicates that bacterial pathogens are controlled in part by innate acting B cells utilizing pattern-recognition receptors and producing granulocyte-macrophage colony-stimulating factor17.\n\nTogether these findings validate the notion that adaptive immune cells can mediate effector functions independent of antigen receptor activation thus serving as innate effectors. Conversely, innate effector cells, NK cells display immunologic memory18, an iconic trait of adaptive immunity. These observations indicate the conventional notion of innate and adaptive cells is in need of revision.",
"appendix": "Author contributions\n\n\n\nBoth authors CFW and CB contributed equally to this article.\n\n\nCompeting interests\n\n\n\nThe authors declare no financial conflict of interest related to this article.\n\n\nGrant information\n\nThis work was supported by grants to CFW from the National Institutes of Health (AI033068 and AI048074).\n\n\nAcknowledgments\n\nThe authors recognize the efforts of the dedicated scientists in their laboratories that contributed to the experimental work including Kirsten Schneider and Antje Rhode, and art design by Vasileios Bekiaris.\n\n\nReferences\n\nSchneider K, Loewendorf A, De Trez C, et al.: Lymphotoxin-mediated crosstalk between B cells and splenic stroma promotes the initial type I interferon response to cytomegalovirus. Cell Host Microbe. 2008; 3 (2): 67–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoseman EA, Iannacone M, Bosurgi L, et al.: B Cell Maintenance of Subcapsular Sinus Macrophages Protects against a Fatal Viral Infection Independent of Adaptive Immunity. Immunity. 2012; 36 (3): 415–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLocksley RM, Killeen N, Lenardo MJ, et al.: The TNF and TNF receptor superfamilies: Integrating mammalian biology. Cell. 2001; 104: 487–501. PubMed Abstract | Publisher Full Text\n\nWare CF: NETWORK COMMUNICATIONS: Lymphotoxins, LIGHT, and TNF. Annu Rev Immunol. 2005; 23: 787–819. PubMed Abstract | Publisher Full Text\n\nBanks TA, Rickert S, Benedict CA, et al.: A lymphotoxin-IFN-beta axis essential for lymphocyte survival revealed during cytomegalovirus infection. J Immunol. 2005; 174 (11): 7217–25. PubMed Abstract\n\nHsu KM, Pratt JR, Akers WJ, et al.: Murine cytomegalovirus displays selective infection of cells within hours after systemic administration. J Gen Virol. 2009; 90 (Pt 1): 33–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIannacone M, Moseman EA, Tonti E, et al.: Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus. Nature. 2010; 465: 1079–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNolte MA, Arens R, Kraus M, et al.: B cells are crucial for both development and maintenance of the splenic marginal zone. J Immunol. 2004; 172 (6): 3620–7. PubMed Abstract\n\nMatsumoto M, Fu YX, Molina H, et al.: Lymphotoxin-alpha-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers. Immunol Rev. 1997; 156: 137–44. PubMed Abstract | Publisher Full Text\n\nKhanna KM, Lefrancois L: B cells, not just for antibody anymore. Immunity. 2012; 36 (3): 315–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHonke N, Shaabani N, Cadeddu G, et al.: Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus. Nat Immunol. 2011; 13 (1): 51–7. PubMed Abstract | Publisher Full Text\n\nZhang D, Zhang DE: Interferon-stimulated gene 15 and the protein ISGylation system. J Interferon Cytokine Res. 2011; 31 (1): 119–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJunt T, Scandella E, Ludewig B, et al.: Form follows function: lymphoid tissue microarchitecture in antimicrobial immune defence. Nat Rev Immunol. 2008; 8 (10): 764–75. PubMed Abstract | Publisher Full Text\n\nRennert PD, Browning JL, Mebius R, et al.: Surface lymphotoxin a/b complex is required for the development of peripheral lymphoid organs. J Exp Med. 1996; 184: 1999–2006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMebius RE: Organogenesis of lymphoid tissues. Nat Rev Immunol. 2003; 3 (4): 292–303. PubMed Abstract | Publisher Full Text\n\nDe Togni P, Goellner J, Ruddle NH, et al.: Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science. 1994; 264: 703–7. PubMed Abstract | Publisher Full Text\n\nRauch PJ, Chudnovskiy A, Robbins CS, et al.: Innate response activator B cells protect against microbial sepsis. Science. 2012; 335 (6068): 597–601. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVivier E, Raulet DH, Moretta A, et al.: Innate or adaptive immunity? The example of natural killer cells. Science. 2011; 331 (6013): 44–9. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "260",
"date": "06 Aug 2012",
"name": "Barry Rouse",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis succinct scholarly review has an interesting story but its title could mislead.Accordingly it gives a nice account about how B cells subserve a protective function other than by their main raison d’etre (antibody production). What it does not do is to provide any evidence for contradictory terminology. Thus the primary function of B cells remains adaptive immunity with their foray into innate immunity also possible, just like countless other cell types. Cells, like us, have many activities some of which are primary and pay the bills and others minor distractions- like surfing if you happen to live in San Diego!",
"responses": []
},
{
"id": "261",
"date": "14 Aug 2012",
"name": "Peter Ghazal",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary summarises recent findings which point to a previously under-appreciated role of B-cells acting as innate effector cells in immunity.The authors build on the notion for a continuum of activity between the innate and adaptive arms of the immune system, integrated at the cellular level via B-cells. Accordingly, this commentary convincingly argues for reconsideration of the conventional view of the role played by innate and adaptive cells of the immune system.",
"responses": []
},
{
"id": "263",
"date": "15 Aug 2012",
"name": "Mariapia Delgi-Esposti",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-8
|
https://f1000research.com/articles/1-7/v1
|
30 Jul 12
|
{
"type": "Research Article",
"title": "High fidelity optogenetic control of individual prefrontal cortical pyramidal neurons in vivo",
"authors": [
"Shinya Nakamura",
"Michael V Baratta",
"Matthew B Pomrenze",
"Samuel D Dolzani",
"Donald C Cooper",
"Shinya Nakamura",
"Michael V Baratta",
"Matthew B Pomrenze",
"Samuel D Dolzani"
],
"abstract": "Precise spatial and temporal manipulation of neural activity in specific genetically defined cell populations is now possible with the advent of optogenetics. The emerging field of optogenetics consists of a set of naturally-occurring and engineered light-sensitive membrane proteins that are able to activate (e.g. channelrhodopsin-2, ChR2) or silence (e.g. halorhodopsin, NpHR) neural activity. Here we demonstrate the technique and the feasibility of using novel adeno-associated viral (AAV) tools to activate (AAV-CaMKllα-ChR2-eYFP) or silence (AAV-CaMKllα-eNpHR3.0-eYFP) neural activity of rat prefrontal cortical prelimbic (PL) pyramidal neurons in vivo. In vivo single unit extracellular recording of ChR2-transduced pyramidal neurons showed that delivery of brief (10 ms) blue (473 nm) light-pulse trains up to 20 Hz via a custom fiber optic-coupled recording electrode (optrode) induced spiking with high fidelity at 20 Hz for the duration of recording (up to two hours in some cases). To silence spontaneously active neurons, we transduced them with the NpHR construct and administered continuous green (532 nm) light to completely inhibit action potential activity for up to 10 seconds with 100% fidelity in most cases. These versatile photosensitive tools, combined with optrode recording methods, provide experimental control over activity of genetically defined neurons and can be used to investigate the functional relationship between neural activity and complex cognitive behavior.",
"keywords": [
"A method for selective and rapid reversible manipulation of neuronal activity is important for parsing out the relationship between prefrontal cortical (PFC) neuronal activity and cognition. Recent optogenetic technologies",
"which allow neurons to respond to specific wavelengths of light with action potential output",
"are now providing a significant advance in our ability to control the activity of select cell populations and neural circuits in behaving animals1. These tools allow for bidirectional control over the neuronal activity2. To date",
"the majority of optogenetic experiments have used transgenic",
"virally-mediated",
"or a combination of the two approaches in mice3–5. Here we use virally-mediated gene delivery of the light-responsive proteins ChR26 or halorhodopsin7 in Sprague-Dawley rat PFC PL pyramidal neurons. Expression of ChR2 and NpHR enables neurons to be depolarized and silenced by pulses of blue and green light",
"respectively. We tested how well PL pyramidal neurons expressing ChR2 followed pulses (10 ms) of blue light delivered at 20 Hz (fidelity). Given the potential importance of long duration light delivery for a variety of protocols we determined if stable responses could be elicited from individual neurons for at least 2 hrs. Lastly",
"we tested green light-induced silencing of spontaneously active NpHR expressing PL neurons to determine if network-driven action potential activity could be silenced continuously for 10 seconds",
"thus establishing the feasibility of long duration silencing for behavioral testing."
],
"content": "Introduction\n\nA method for selective and rapid reversible manipulation of neuronal activity is important for parsing out the relationship between prefrontal cortical (PFC) neuronal activity and cognition. Recent optogenetic technologies, which allow neurons to respond to specific wavelengths of light with action potential output, are now providing a significant advance in our ability to control the activity of select cell populations and neural circuits in behaving animals1. These tools allow for bidirectional control over the neuronal activity2. To date, the majority of optogenetic experiments have used transgenic, virally-mediated, or a combination of the two approaches in mice3–5. Here we use virally-mediated gene delivery of the light-responsive proteins ChR26 or halorhodopsin7 in Sprague-Dawley rat PFC PL pyramidal neurons. Expression of ChR2 and NpHR enables neurons to be depolarized and silenced by pulses of blue and green light, respectively. We tested how well PL pyramidal neurons expressing ChR2 followed pulses (10 ms) of blue light delivered at 20 Hz (fidelity). Given the potential importance of long duration light delivery for a variety of protocols we determined if stable responses could be elicited from individual neurons for at least 2 hrs. Lastly, we tested green light-induced silencing of spontaneously active NpHR expressing PL neurons to determine if network-driven action potential activity could be silenced continuously for 10 seconds, thus establishing the feasibility of long duration silencing for behavioral testing.\n\n\nMaterials and methods\n\nSubjects. Male Sprague-Dawley rats (6–8 weeks) were housed in pairs on a 12-h light/dark cycle. Rats were allowed to acclimate to colony conditions for 7–10 days prior to surgery. All animal procedures were approved by the Institutional Animal Care and Use Committee of University of Colorado at Boulder.\n\nVirus injection. An AAV vector carrying the opsin gene encoding the light-gated nonselective cation channel ChR2 or the light-driven third generation chloride pump NpHR under the control of the excitatory neuron-specific promoter CaMKllα (AAV-CaMKllα-ChR2-eYFP or AAV-CaMKllα-eNpHR3.0-eYFP) was injected into the PL (A/P: +2.7 mm; M/L: ±0.5; D/V: -2.2 mm) using a 10 μl syringe and a thin 31 gauge metal needle with a beveled tip (Hamilton Company). The total injection volume (1 μl) and rate (0.1 μl/min) were controlled with a microinjection pump (UMP3-1, World Precision Instruments). The virus titer was 3 × 1012 particles/ml.\n\nFor simultaneous extracellular recording and light delivery, we developed a custom-made optrode consisting of a tungsten electrode (1~1.5 MΩ, MicroProbes) attached to an optical fiber (200 μm core diameter, 0.48 NA, Thorlabs). First, a tungsten electrode was attached to a glass capillary tube. An optical fiber was then inserted into the capillary tube and fixed loosely to the electrode using suture thread. Position of the fiber tip was adjusted so that the center-to-center distance between the electrode tip and the fiber tip was ~300 μm.\n\nEach rat transduced in the PL with ChR2 or NpHR was anesthetized with urethane (1.5 g/kg, i.p.), and a small hole was made on the skull above the PL. An optrode was then carefully lowered through the hole until emergence of optically evoked or inhibited signals. Recorded signals were band-pass filtered (0.3–8 kHz), amplified (ExAmp-20K, Kation Scientific), digitized at 20 kHz (NI USB-6009, National Instruments), and stored in personal computer using custom software (LabVIEW, National Instruments). The custom software was also used for controlling a blue (473 nm) or green (532 nm) single diode laser (Shanghai Laser & Optics Century Co.).\n\n\nResults\n\nTo test the functionality of ChR2-transduced PL, we performed in vivo extracellular recordings from PL neurons during delivery of blue light. Indeed, PL neurons showed spiking with perfect fidelity by the blue-light pulse trains at various frequencies (1–20 Hz) in ChR2-transduced PL (n=8, Figure 1a).\n\na) Photoactivation of ChR2-transduced PL neurons at various frequencies (1–20 Hz). b) Voltage trace showing blue (473 nm) light-evoked spiking of PL neuron to 20 Hz delivery of 10 ms blue light pulses. Inset: representative light-evoked single-unit response. c) Averaged response of this neuron to 10 ms light pulse calculated from all events in above trace (bin width of 1 ms).\n\nFigures 1b and 1c show the results of 10 ms blue-light pulse delivery (<250 mW/mm2) at 20 Hz for 5 seconds (100 pulses total). Even when using this longer duration protocol, each light pulse was able to evoke an action potential with high reproducibility. We then recorded 120 minutes (2 hrs) of light-evoked responses in PL neurons (n=2).\n\nThe average spontaneous firing rate during the baseline period (5 seconds) during the first 10 minutes of recording was 0.68 +/- 0.10 (Cell 1) and 1.13 +/- 0.19 (Cell 2). Five seconds after delivery of 100 10 ms blue light pulses at 20 Hz the average spontaneous firing rate was 0.56 +/- 0.13 (Cell 1) and 0.067 +/- 0.067 (Cell 2). The average spontaneous firing rate during the baseline period during the last 10 minutes of the 120 minute recording was 0.12 +/- 0.08 (Cell 1) and 1.83 +/- 0.23 (Cell 2) and after blue light delivery the average firing rate was 0.16 +/- 0.074 (Cell 1) and 0.4 +/- 0.15 (Cell 2).\n\nAfter a baseline period of 5 seconds, trains of 10 ms light pulses (100 pulses total) were repeatedly delivered at 20 Hz with an inter-train interval of 2 min for 120 minutes (Figure 2). Figure 2b shows the high fidelity responsiveness to the light train across the 2 hr recording. Mean spike probabilities (± standard error of the mean) in response to each of the 100 light pulses delivered at 20 Hz for first and last 10 min recording session were as follows: 1.04 ± 0.020 (Cell 1, first) and 0.91 ± 0.034 (Cell 1, last); 2.16 ± 0.055 (Cell 2, first) and 2.19 ± 0.056 (Cell 2, last, Figure 2b).\n\na) Voltage traces of the light-evoked spiking acquired at the time point of 0, 60 and 120 min after the beginning of recordings. b) Raster plot showing all 61 repetitions (2 min interval, 120 min total) of the light-induced activation. c) Average firing rate (thick line) with standard error of the mean (SEM, thin line) calculated from raster plot above (bin width of 100 ms).\n\nWe then tested in vivo photoinhibition of spontaneous activity in NpHR-transduced PL.\n\nFigure 3 shows the results of NpHR-induced inhibition of PL neuronal activity using 10 seconds of continuous green light exposure (<250 mW/mm2). In contrast to the ChR2 results, we observed silencing of spontaneous activity in the PL during light delivery (n=7) compared to pre- and post-light periods. Only four (13%, from two cells) out of a total of 30 trials had break through action potentials during the period of 10 second laser illumination (e.g. Figure 3b). To functionally characterize the neuronal activity we constructed autocorrelation histograms (ACH) and calculated the average firing rate during pre- and post-laser period for each neuron (Figure 4). The ACH revealed two types of neurons that were functionally classified into either phasic or non-phasic firing. Phasic neurons showed correlated activity marked by peaks in their ACH (Figure 4a), indicating their periodic network-driven firing pattern, but non-phasic neurons showed no correlated activity (Figure 4b). Two out of three phasic neurons showed a rebound increase in excitability after the light illumination (Figure 4c), but overall there appeared to be no change (Figures 4c and d). Further recordings are necessary to determine if continuous activation of NpHR with green light leads to some form of plasticity depending on their type of neuronal inputs. Control recordings using blue and green light (light intensities of 250 mW/mm2) did not alter spontaneous firing rates in PL neurons that did not express either ChR2 or NpHR as inferred from the lack of time-locked light responses (data not shown), which suggests no unexpected off-target effects of light alone on neuronal activity.\n\na) Representative trace of spontaneous activity of a NpHR-transduced PL neuron that was inhibited by the continuous green (532 nm) laser delivery. b) Raster plot showing five repetitions of the light-induced silencing in this neuron. Each unit activity is plotted as a dot. c) Average firing rate (thick line) with SEM (thin line) calculated from these five repetitions (bin width of 1000 ms).\n\na, b) Auto-correlation histograms (ACH) for each recorded neuron showing periodic firing pattern in phasic neurons, but not in non-phasic neurons. ACH were created by summing the number of intervals between non-consecutive events as well as intervals between consecutive events (bin width of 20 ms). c, d) Average firing rate of each neuron during pre- and post-laser period. Note that the result of one neuron is not shown because of the low number of spikes.\n\n\nDiscussion\n\nIn this study we demonstrated high fidelity in vivo recording of individual PL pyramidal neurons transduced with ChR2 or NpHR. Single-unit responses were recorded in response to trains of 10 ms blue light pulses up to 20 Hz with a mode of activation onset time of 7 ms. Light delivered through a custom optrode (<250 mW/mm2) induced stable and robust activation that persisted for the duration of recording (2 hrs in some cases).\n\nTo understand how neuronal activity influences behavior it is necessary to not only activate, but also silence neuronal activity with precision. We used 5–10 seconds of green light (<250 mW/mm2) to silence spontaneously active phasic and nonphasic firing NpHR transduced PL pyramidal neurons. Both phasic and nonphasic pyramidal neurons were potently inhibited for the duration of light delivery (up to 10 seconds). In some phasic firing neurons we observed a slight rebound increase in excitability upon termination of light. More recordings are necessary to determine the mechanism for rebound excitability or if it is particular for phasic firing neurons. A transient increase in activity shortly after termination of light-induced hyperpolarization (~100 ms) as seen in Figure 3C would be consistent with activation of a hyperpolarization activated (Ih) inward current, while more persistent increases in activity may indicate indirect effects resulting from activation of the NpHR Cl- pumps and the resulting change in extracellular Cl-. Recently, Raimondo et al. (2012) reported that in vitro synaptically-evoked spike probability significantly increases shortly after termination of photoinhibition in NpHR-expressing hippocampal neurons8. Our results are the first to suggest this phenomenon may happen in vivo to a subpopulation of phasic firing cells in the PL. Future experiments are necessary to understand the relationship between neuronal activation/silencing on behavior and the possible off target effects of ChR2 or NpHR activation.\n\nThe technique of in vivo light delivery and simultaneous recording of neuronal responses holds the promise of establishing direct causal relationships between the onset/offset and pattern of activity in specific genetically-determined neuronal populations and corresponding time-locked behavioral events. Our results establish the feasibility of long duration high fidelity activation or continuous silencing of individual neurons for a variety of behavioral experiments.",
"appendix": "Author contributions\n\n\n\nS.N., M.V.B, and D.C.C. designed experiments, analyzed data and wrote the paper. S.N., M.V.B., M.B.P., and S.D.D. carried out experiments. M.V.B. and M.B.P. assisted with molecular biology and virus preparation.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests declared.\n\n\nGrant information\n\nThis work was supported by National Institute on Drug Abuse grant R01-DA24040 (to D.C.C.), NIDA K award K-01DA017750 (to D.C.C.).\n\n\nReferences\n\nBernstein JG, Boyden ES: Optogenetic tools for analyzing the neural circuits of behavior. Trends Cogn Sci. 2011; 15(12): 592–600. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaratta MV, Nakamura S, Dobelis P, et al.: Optogenetic control of genetically-targeted pyramidal neuron activity in prefrontal cortex. Nat Precedings. (posted Apr 2, 2012). Publisher Full Text\n\nTye KM, Prakash R, Kim SY, et al.: Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature. 2011; 471(7338): 358–362. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao S, Ting JT, Atallah HE, et al.: Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat Methods. 2011; 8(9): 745–752. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWitten IB, Lin SC, Brodsky M, et al.: Cholinergic interneurons control local circuit activity and cocaine conditioning. Science. 2010; 330(6011): 1677–1681. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoyden ES, Zhang F, Bamberg E, et al.: Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci. 2005; 8(9): 1263–1268. PubMed Abstract | Publisher Full Text\n\nZhang F, Wang LP, Brauner M, et al.: Multimodal fast optical interrogation of neural circuitry. Nature. 2007; 446(7136): 633–639. PubMed Abstract | Publisher Full Text\n\nRaimondo JV, Kay L, Ellender TJ, et al.: Optogenetic silencing strategies differ in their effects on inhibitory synaptic transmission. Nat Neurosci. 2012; 15(8): 1102–1104. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "264",
"date": "31 Jul 2012",
"name": "Howard Eichenbaum",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful confirmation of the optogentic control of neural activity in the rat prefrontal cortex.The basic effectiveness and characterization of channel rhodopsin and halorhodopsin have been reported multiple times, so this study mainly extends the same findings to a new area of cortex in the anesthetized rat. The study design is straightforward, the results are well described, and the findings mainly solid. So, if novelty is a low priority and the hottest new technologies (in this case optical control) are the highest priority, I recommend publication.The authors should provide a more comprehensive report of previous findings in other preparations and compare results, particularly with regard to the rebound effects. Also, shouldn’t they run a light only (no virus) control to show that light itself does not drive or inhibit neuronal activity?The distinction between phasic and not is fuzzy. What exactly is phasic about the phasic cells? They should provide a quantitative characterization.The low baseline and evoked response rates are concerning. Is this the anesthesia?",
"responses": [
{
"c_id": "59",
"date": "03 Aug 2012",
"name": "Donald Cooper",
"role": "Author Response",
"response": "We thank Dr. Eichenbaum for his helpful critique of our research article. One goal we had for this research article posted in the new F1000Research science publishing model was to quickly share some of our ongoing recording datasets in order to encourage collaboration with others in the field. Our goal is to ultimately publish these datasets together with more complete analysis and conclusion and other work from our lab or new collaborators. Because we intend on publishing this data with more data and more complete analysis and conclusions we kept the analysis and conclusions to a minimum so as not to violate subsequent journal publishing policies regarding prior publication.The question of novelty:As was pointed out this is not the first demonstration of light-evoked activation or silencing of neural activity, so in that regard we have only successfully replicated others. However, we presented several novel findings that have not yet been reported in the literature. For example, we present novel data showing that channelrhodopsin continues to function even after 2 hrs of recording of an individual virally infected neuron. We demonstrated that CaMKII expressing (light responsive) pyramidal neurons can be classified into phasic and nonphasic based on the periodicity revealed in the autocorrelation histogram of their firing pattern and this too is novel. Overall, we described a novel protocol demonstrating light activation across a range of frequencies that may be relevant for behavioral procedures. Anyone interested in the light-evoked neural response parameters for behavioral testing can be assured that under the conditions we described high fidelity light responsiveness will be maintained. Despite these novel findings, novelty is not a criteria for the F1000Research publishing model. In fact, replication is encouraged (see http://f1000research.com/about/).Regarding control recordings using light alone:It was suggested that we try recording from uninfected neurons to see if light alone might drive action potential firing. We have done this several times and in the article we stated the following, “Control recordings using blue and green light (light intensities of 250 mW/mm2) did not alter spontaneous firing rates in PL neurons that did not express either ChR2 or NpHR as inferred from the lack of time-locked light responses (data not shown), which suggests no unexpected off-target effects of light alone on neuronal activity.” This data will be provided in future publications along with more analysis and conclusions.Regarding a more comprehensive discussion of related work and comparisons of rebound responses:We certainly intend on following this advice in future journal submissions. For this research article we do not have enough data yet to make any conclusions about whether the rebound phenomenon following halorhodopsin that has been reported in vitro actually extends to the in vivo milieu. Our data is suggestive and as we stated in the article more data is needed and these experiments are ongoing.Low baseline firing:The baseline firing is typical for in vivo prefrontal cortical pyramidal neuron spontaneous firing under anesthetized conditions (e.g. Laviolette and Grace, 2006). The light evoked firing was done in neurons that were selected for their sparse firing rate in order to better show the ability of light-activated channelrhodopsin to initiate action potential activity. For silencing, we explicitly chose neurons with spontaneous activity so that we could observe the silencing effect of halorhodopsin.We are excited to contribute the the new model of science publishing F1000Research has offered and we appreciate the thoughtful comments from Dr. Eichenbaum that will improve our subsequent publication. We also welcome new collaborators resulting from this research article!"
}
]
},
{
"id": "262",
"date": "31 Jul 2012",
"name": "Ege Kavalali",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this is a well-executed study that fits the publication objectives of F1000Research.The authors report in vivo single unit recordings from cortical prelimbic pyramidal neurons expressing channelrhodopsin-2 or halorhodopsin. Although the basic premise of this article is not entirely novel and the authors refrain from arriving at any firm biological conclusions, the results raise some interesting issues. For instance, in Figure 1 and 2 channelrhodopsin-2 induced firing shows a rapid decrease in frequency (Figure 1b and 2c) which can elicit significant plasticity of the neurotransmitter release output of these neurons. Along the same lines in Figure 3, after 10 sec of green light exposure the firing frequency recovery shows significant rebound facilitation, which again could significantly impact neurotransmitter release output of these neurons. Therefore, in order to be able interpret the behavioral correlates of these activity patterns accurately, one would need to know the synaptic output properties of these neurons during and after optogenetic stimulation/silencing.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-7
|
https://f1000research.com/articles/1-5/v1
|
20 Jul 12
|
{
"type": "Commentary",
"title": "NIH Portfolio Allocation, Lemmings, and the Silent Spring: A Time-Capsule Commentary & Its Update",
"authors": [
"Mark Boothby"
],
"abstract": "With the release of the US President’s proposed budget for the Federal Fiscal year (FY) 2013, to start October 1, 2012, we’ve spun yet again into the mad vortex of an appropriation season. Fundamental re-thinks of how biological and medical research are prioritized and funded are urgently needed, but sadly appear to be unlikely unless the research and advocacy communities push harder and in a more unified manner. Early in the Obama presidency and the NIH Directorship of Dr Francis Collins, the FASEB Office of Public Affairs performed an analysis of trends in funding of R01 and other Research Project Grants and shared that with the Director and his office. Using the FASEB analysis, whose numbers drew on NIH data, an independent commentary (below) was submitted to (but not published in) Science. With the analysis a few years old, this older viewpoint is followed by updates that touch on how the trends have fared since early 2010 and comment on other aspects of the ongoing cull in biomedical research. In particular, data on some of the growth areas that continue to prosper at the expense of the ever-declining direct support for R01 science are discussed.",
"keywords": [
"Some milestones take on particular resonance and prompt reviews of trends – a New Decade",
"the first 200 days of a new National Institutes of Health (NIH) Director",
"and a Policy Forum from the Director1. The long Congressional appropriation season is opening",
"and for the first time in recent memory the President has proposed a budget in which the NIH might lose less ground to inflation. Accordingly",
"it is timely to open a discourse regarding changes in management of the NIH enterprise over the past 8–10 years. The data (Figure 1 and Figure 2) should catalyze an analysis of the premises and likely impact of these changes on health-related research",
"education and progress in the USA. The multitude of productive research teams and themes extinguished in recent years suggests the view that trends in biomedical research funding allocations are changes analogous to those heralded in \"Silent Spring\"2. This little-discussed toll should be a part of analyzing how the US is faring in its need to stay the locomotive of discovery and progress in health."
],
"content": "Lemmings, and the Silent Spring [Feb. 2010]\n\nSome milestones take on particular resonance and prompt reviews of trends – a New Decade; the first 200 days of a new National Institutes of Health (NIH) Director, and a Policy Forum from the Director1. The long Congressional appropriation season is opening, and for the first time in recent memory the President has proposed a budget in which the NIH might lose less ground to inflation. Accordingly, it is timely to open a discourse regarding changes in management of the NIH enterprise over the past 8–10 years. The data (Figure 1 and Figure 2) should catalyze an analysis of the premises and likely impact of these changes on health-related research, education and progress in the USA. The multitude of productive research teams and themes extinguished in recent years suggests the view that trends in biomedical research funding allocations are changes analogous to those heralded in \"Silent Spring\"2. This little-discussed toll should be a part of analyzing how the US is faring in its need to stay the locomotive of discovery and progress in health.\n\nTotal NIH spending on RPGs (P01, P42, PN1, U01, U19, UC1, NIGMS P41, and all R-series) in each Federal Fiscal Year (FY) divided by total NIH budget. Analysis courtesy of FASEB, 2009, drawing on data from NIH Data Book.\n\nTotal NIH spending on R01-equivalent grants (including the R29 program in earlier FY) in each Federal Fiscal Year (FY) divided by total NIH budget. Inset numbers highlight total numbers of R01 awards in ‘active’ status (a term that includes no-cost extensions in time) at the beginning of the post-doubling period and the last FY prior to ARRA. Analysis courtesy of FASEB (2009), using data from NIH Data Book.\n\nThe force and persistence of the underlying trends emerged in data that capture overviews of directions the NIH has taken. They include the last federal fiscal years (FY) that can reliably picture management trends of the NIH prior to the short-term impact of the American Reinvestment and Recovery Act (ARRA). A few data snapshots bear particular comment. Damage continues to worsen as the NIH appropriation loses ground to inflation year after year in the \"post-doubling era\"3. However, it is equally true that the NIH decreased its allocation of funds to the gold standard of investigator-initiated research (the unsolicited R01 grant) by $300,000,000 for FY2008 despite an overall budget $1.5 billion higher than in FY2004. As such, a rising tide seems to have been scuttling the boat rather than lifting it. The full post-doubling era (FY2003–2008) does not offer a much more encouraging picture – the NIH budget increased by $2.8 billion, and only 3% of that increase was allocated to the R01 program.\n\nThese and other changes at NIH appear to embody a philosophy during the past 8–10 years that, in the present era, the foundation of successfully advancing human health has somehow changed and the rules are different. This view culturally resembles thoughts about business and finance during the same era: ‘now things are different’, i.e., advances and the frontier of new investment tools meant that the fundamental rules of finance and risk had been profoundly changed. Analogously, the evolving NIH philosophy appears now to tilt more toward larger programs, central management, and central direction as a paradigm based on the premise that ‘the rules are different’ now compared to earlier days, and that ‘innovation’ or ‘translation’ didn’t occur under investigator-initiated mechanisms or ‘the old way’.\n\nThe data represent a significant turn away from a cornerstone of the NIH during its long period of success. The first question asked by working scientists after viewing these data (striking declines in RPGs as a whole, and even more strikingly in funding of R01s, the bedrock of NIH success) is \"So where has all that money been going?\" Different scientists seem to have best guesses (most commonly, large contracts and centers managed centrally by the NIH), but a striking thing is that these are guesses: the answer is not readily apparent despite a statutory mandate for greater transparency. To provide a rationale for such a large shift in portfolio allocation, a key question in management planning of each independent Institute must be: \"What is the evidence of disproportionate success from this shift of funding focus?\"\n\nHistorically, the primacy of the Research Project Grant and the unsolicited R01 were central to a research culture that promoted innovation and creativity from the widest and most diverse range of scientists possible. This tradition was, and remains, absolutely vital. Great medical advances stem from scientists who originally were not at the top of the food chain working on questions that would originally not have been considered among the ‘hot areas’ before years of groundwork followed by a breakthrough discovery. Peer review on its surface may seem to engender conservatism because study sections take their role as guardians of precious taxpayer dollars seriously, but R01 grants embody key features essential for innovative discoveries and long-term success in approaching the unknown. Vital qualities of the R01 grant that drive this sort of progress are (i) the awards are renewable, multi-year grants of sufficient duration and (ii) flexibility in allowing adjustments of direction. Especially when unsolicited, the duration and flexibility encourage innovation and harness the creativity of tens of thousands of investigators with ideas that current paradigms or central planners would not likely imagine, let alone prioritize. In short, R01 science promotes innovation.\n\nAt the same time, a sufficiently large and diverse portfolio is vital for \"consolidation\", i.e., the validation and incremental extension or solidification of apparent discoveries. This part of science is as integral to progress as innovation but gets short shrift by implication. To paraphrase Thomas Edison, one of the most successful inventors and innovators in US history, progress is \"1% innovation and 99% perspiration\". A balance is needed between innovation (or ‘transformation’) and ‘consolidation’ in advancing human health by ‘reducing innovation to successful practice’ by validation and extension (i.e., consolidation). Will a NIH moving to centrally managed large contracts and large infrastructure projects – with innovation in a fringe minority of its funding and an ever-dwindling amount of consolidation – be the optimal model for advancing health? Similarly, shifting funds to translation raises the urgent question: who but the NIH will maintain the US’ lead in basic investigation, and how deep will the understanding of mechanisms underpinning translated ideas be? The result of the previous cultural period is a proven success. Whether the changes at the NIH might improve on that record is a central issue for skepticism and open discussion.\n\nDespite much-discussed and glaring shortcomings in the delivery and quality of medical treatment in the US, we have witnessed an extraordinary fall in death rates from ischemic and atherosclerotic vascular diseases and, for all the concern that progress is not even greater, major advances in the fight against cancers. In the context of a paradigm shift for the NIH, there is a need for broad-based input addressing hard questions. Foremost among these is whether diverting money from unsolicited research project grants and R01s into large-scale programs that ostensibly will ‘de-risk’ agents for pharma or hew to 5 year plans for identification of new therapeutics is realistic or viable. The continuing force of Vannevar Bush’s germinal report rings as true now as through the past 65 years:\n\n\"The striking advances in medicine [. . .] have been possible only because we had a large backlog of scientific data accumulated through basic research in many scientific fields\", \"Scientific progress on a broad front results from the free play of free intellects, working on subjects of their own choice, in the manner dictated by their curiosity for exploration of the unknown. Freedom of inquiry must be preserved under any plan for Government support of science\", and \"Basic research is a long-term process – it ceases to be basic if immediate results are expected on short-term support. Methods should therefore be found which will permit the agency to make commitments of funds from current appropriations for programs of five years duration or longer.\"\n\nWith this latter point in mind, one striking trend within the R (investigator-initiated) series of NIH grants is the trend towards ever-larger numbers of R21 (2 yr) awards as the number of R01-equivalent grants falls.\n\nThe mission of the NIH and its great opportunities and promise were beautifully summarized by the NIH Director but two items were notable in the Director’s Research Agenda1. First, while observing that creative insights of individual investigators are the foundation of success in advancing human health, it states that ‘increasingly investigators are working in teams’ and ‘a careful balance is needed between individual investigator-initiated projects and large-scale community resource-generating efforts’. In fact, investigators in an ‘R01- (& P01-) centric’ era of NIH culture involved teams of investigators and the NIH allocation of funds always involved a careful and judicious balance of large projects, large multi-center clinical science, and other team or infrastructure investigations. The questions for NIH and its Institutes are: what is the appropriate balance; why has the balance been changed at present; and, since a shift went on for 8–10 years, what evidence is there in terms of rigorous, independent ‘cost-benefit’ analyses to support continuing the change in allocation?\n\nThe most urgent priority, and most vitally needed remedy for these potentially devastating effects on the biomedical research, education and training enterprise, is for Congress to sustain and expand increases in funding of the NIH. However, appropriations in the post-doubling era have been framed in a broader fiscal picture. The Federal fiscal picture for years to come will look worse than it did during 2000–2009. The NIH appropriation for this fiscal year and the President’s proposed budget – with a 3.2% increase and a pie chart suggesting 53.5% of the appropriation may go to RPGs – might be a cause for some optimism unless the rising tide continues to scuttle the RPG/R01 boat. For instance, the proposed impact from a $1,000,000,000 increase would be a drop in the pool of money for new/competing renewal RPGs (200 less new grants for what is termed a high priority). A comparison of the numbers for RPGs to the period 2004–2008 is bleaker, and data trends tracking the fraction of RPG money going to R01-equivalent awards suggests an even more dire prognosis. NIH and the health research community need a better plan for how to deal with increases less than what is genuinely needed, but more than the cuts that so many government agencies and individual citizens face. A central issue for such planning is to discuss the question whether the disproportionate cuts in the RPG and R01 programs are wise choices. Alternatively, is this the time for tough choices be made to move back toward the portfolio allocations that prevailed as NIH’s norm in its period of successes proven to advance human health?\n\nAt present, large swaths of the research community are in a state akin to an infection at the stage when it is controlled but approaching the point of tipping into systemic shock and irreversible organ damage. In weighing costs and benefits of the large change in portfolio allocation at NIH (i.e., progressively underweighting the R01 component), the long-term effects on US health are little discussed. Part of what drove the very best in American medical training was the involvement of a large set of active researchers in medical training at all levels. Set-backs to training medical professionals in the advanced science of health will be set-backs to progress in health itself. The damage to that culture needs to be reckoned among the secondary consequences of the NIH shifts even if the statutory mission of NIH does not include direct support of medical or health education other than the NRSA program. As primary damage, what looms is a ‘cull’ eliminating further thousands of trained team leaders (faculty active in research and education) and unique biomedical research avenues with important contributions hanging in the balance. Perhaps witnessing this cull of established research teams will inspire talented and more innovative minds to flock to biomedical research careers?\n\nSadly, in light of these trends the impression many active, talented, productive mid-career biomedical researchers have formed as to how their activities are viewed by the NIH is that lemmings offer the best metaphor. This species is subject to explosive population growth and sudden, devastating declines. While enshrined in popular imagination as committing mass suicide by jumping off a precipice, in fact the lemmings were actively pushed off the edge to make a film. NIH funding will always be limited relative to the opportunities, but great opportunities are best realized by an optimal portfolio allocation. For the long-term health of the country, let’s hope that cohorts of scientists won’t be herded off \"The Cliff.\"\n\n\nTwo years later – an update [Feb. 2012]\n\nA first question to consider in relation to the original analysis is about the data accuracy. In mid-2010, the NIH Office of Extramural Research (OER) kindly provided its analysis of the numbers (Figure 3 and Figure 4, scans of 2010 OER data labeled as Figure 2 and Figure 4). While apparently accounting for some monies (SBIR/STTR) a bit differently, the basic trends were similar to the FASEB analysis (for instance, a drop in R01-funded research from around 42.5–43% of total NIH budget in the 15 year period FY1988–2002, to 38% in FY 2009). What dynamic drives the sort of change in portfolio allocation identified in these data? One thoughtful angle on why so many of the Institutes and Centers of the NIH trend in this way was spelled out in late 2011 in a marvelous opinion piece in Genome Biology4.\n\nTotal NIH spending on RPGs (P01, P42, PN1, U01, U19, UC1, NIGMS P41, and all R-series; green bars) as well as other components of the research funding enterprise, for each Federal Fiscal Year (FY) 1983 to 2009, divided by total NIH budget. Analysis courtesy of NIH OER (abstracted from a July, 2010 letter replying to input).\n\nTotal NIH spending on various components of the RPG pool (P01, P42, PN1, U01, U19, UC1, NIGMS P41, and all R-series) for each Federal Fiscal Year (FY) 1983 to 2009, expressed as a % of the overall (but shrinking) RPG allocation. R01, green bars. Analysis courtesy of NIH OER (abstracted from a July, 2010 letter replying to input).\n\nIn the spirit of Congressional report language in NIH appropriations, the OER had several enlightening updates as part of soliciting blog-style input. Congress correctly indicated that constantly whittling down award sizes and steadily decreasing the numbers of grant awards does not provide a recipe for success. Moreover, the troll lurking under the bridge for FY2013 is that, with the predictable failure of the Gang of Twelve (\"Joint Select Committee on Deficit Reduction\"), the NIH faces the possibility of a sequestration that would cut its budget by over 7.5%. NIH OER offered some insights into ways to model how much various changes could help, framed in an online solicitation, \"How Do You Think We Should Manage Science in Fiscally Challenging Times?\" One of the most striking points of data was that the probability of success on applications increased as application sizes get larger.\n\nMore recently, OER provided further data that support the Lemming Theory of Biomedical Research(er) Management. This thoughtful piece is noteworthy in documenting that just for one year, FY2010 to FY2011, the number of new/competing Research Project Grant awards dropped from 9432 (not including ARRA awards, which have now gone away) to 8776. That fall represents a decline of about 7% in just one year, and of course the end of an ARRA means even more projects consigned to the ashbin! Since NIH reports that, within the extramurally funded community, about 80% of funded PIs lead only one RPG, it is likely that 676 ‘lost RPGs’ represent a sizable cull in the PI community.\n\nThese points bring one back to the President’s proposed FY2013 budget. The budget as a whole has virtually no chance of being adopted by Congress – especially as tough re-election campaigns are in gear for the President and Congress, with fiscal and economic issues as major fronts in the war ahead. Nonetheless, the NIH is an agency managed by the Executive Branch so the budget proposal reflects a mindset that will continue to prevail – barring sufficient pressure to drive change.\n\nA brief pause is in order to think happy thoughts. If one forgets about the possibility of sequestration, the budget at least requests no decrease. Many other agencies will be cut. That said, the executive summary tips a glance at the cards in noting that the pool of money for RPGs should go down (albeit a mere $26,000,000 – aka ~50 10-module R01 grants) while continuing cost escalation in R&D contracts (up $108,000,000) and the NIH intramural research program (up $22,000,000). That the request continues the trend noted in the 2009 FASEB analysis requires looking at the numbers. Strikingly, money for the RPG is down to 51% of NIH total funding (closer to 54% if one includes SBIR/STTR). Even with the shaky assumption that the R01 will hold its ground at 69% of RPGs (FY2009, down from ~75+% in the glory days of the NIH), this budget would drop the R01 below 37% of the overall NIH budget (down from ~43% in the FY1988–2002 period). Fear not, however! Despite the drop in requested monies, \"The number of new or competing RPGs would increase by 672, resulting in an estimate[d] success rate of 19 percent\". Truly, we live in magical times!*\n\nSeriously folks, the bottom line on how the administration proposes to achieve this increase is to cut awards in their \"off-years\" (non-competing continuation awards), laudably to cut out the preferential treatment of non-modular awards (i.e., eliminate the cost-escalations that they, but not non-modular awards, receive), and push the mix toward smaller and shorter Research Project Grant awards. Incidentally, a good by-product of the OER piece is that it gives some clues on ways to use the NIH RePORTER tool to get at some of the data on one’s own. For the key insights into trends after breaking total costs down into direct vs. indirect cost components, and extramural vs. intramural funding, the NIH budget text is the place to go for further gems. For instance, taking the extramural funding pie as a whole, the data show that the aggregate trend in Facilities & Administration (F&A, or \"indirect\") costs continues a long-suspected climb. Sure, these costs have \"only\" increased their share from ~37.2% (FY2002 & 3) to 39% (FY2011). And, sure, the intramural program has \"only\" climbed 0.6% in its share of the overall NIH budget (10.4 to 11%, which on a $30 billion base means $180,000,000). But 1% of a $22 billion extramural research effort is $220,000,000, or ≥ 500 more 10-module R01 grants. Add to that the money escalating R&D contracts but not basic science research, and one arrives at cost escalations of as much as $725,000,000 in a post-doubling era of \"flat funding\", which have to come at the expense of direct funding for R01 science.",
"appendix": "Competing interests\n\n\n\nNo relevant competing interests declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nTyrone Spady of the FASEB Office of Public Affairs conducted data analyses and initial graphing for Figure 1 and Figure 2, with supervision and input from Howard Garrison (head of FASEB Office of Public Affairs), and some input from Mark Boothby. Figure 3 and Figure 4 represent graphs prepared on behalf of \"The People\" by Dr Sally Rockey of the Office of Extramural Research NIH (DHHS, USA).\n\n\nReferences\n\nCollins FS: Research agenda. Opportunities for research and NIH. Science 2010; 327: 36–37. PubMed Abstract | Publisher Full Text\n\nCarson R: Silent Spring (Boston: Houghton Mifflin, 1962). Reference Source\n\nKorn D, Rich RR, Garrison HH, et al.: Science policy. The NIH Budget in the post-doubling era. Science 2002; 296: 1401–2. PubMed Abstract | Publisher Full Text\n\nPetsko GA: Dominoes. Genome Biology 2011; 12: 134. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "251",
"date": "31 Jul 2012",
"name": "Sebastian Fugmann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Commentary by Mark Boothby adds to the increasingly louder voices expressing their concerns about the trajectory of NIH-funded biomedical research. It takes the plain numbers and places them in the context of the day-to-day reality in a R01-based research group which, as the title suggests, might be a species going extinct.While some critics might portrait this manuscript as a complete exaggeration and an unwarranted “the end of the world is near” song, the underlying facts and numbers are quite clear – the number of old-school R01-supported labs is slowly but surely shrinking. Allocation of public funds for scientific research is a political decision, and as such they are a compromise between opposing force that try to achieve their very own agenda – on the one side the supporters of large goal-oriented research programs (largely the powerful leaders of such huge and expensive projects – while many scientist would love to be such leader, very few would simply dream of being only a tiny little almost dispensable piece of such big scientific machinery) on the other hand the traditional principal investigator who follows his own intuition and fights every five years to get his R01 renewed to continue his work. Thus lobbying to reconsider the ongoing changes is a legitimate action, and this commentary spells out what has been a major topic of conversations during coffee breaks at scientific conferences and grant study sections.One question that remains, however, is whether the arguments of scientists have a stronger impact when presented in a somewhat dull and sober style like a traditional research paper, or as it is done here in a more accessible fashion, including a healthy dose of cynicism. Clearly, this is up to the reader to decide, but even if one does not like the tone, hopefully some of the message will stick.",
"responses": []
},
{
"id": "254",
"date": "31 Jul 2012",
"name": "Nina Papavasiliou",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis well documented commentary goes hand in hand with the very recently published Science article (much shorter by necessity). Both pieces are raising a RED alert on the significant and PRESENT danger of the whole NIH system, which has been significantly degraded during the last decade, now literally going off the cliff.Clearly, a major reorganization is in order, and this will happen by necessity, with or without the input of working scientists. To make an analogy to current affairs, the federally funded scientific enterprise in the USA is functionally bankrupt, and the only choice remaining is between an orderly reorganization, or, a disorderly collapse. Let’s hope for the former.",
"responses": [
{
"c_id": "55",
"date": "08 Aug 2012",
"name": "Horia Georgescu",
"role": "Reader Comment",
"response": "Or, a disorderly reorganization (the “voie royale” in science, however)."
}
]
},
{
"id": "255",
"date": "02 Aug 2012",
"name": "Alan Schechter",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary on NIH budgeting is a cri de coeur from a well-respected MD/PhD immunology investigator about his perception of the untold damages that recent changes in NIH budgeting will likely have on “the long-term health of the country”.His views, which are likely representative of a great many NIH-supported scientists, appear to focus primarily on the cyclic changes in dollars of support leading to continued “cull[ing] of established research teams” (i.e., the lemmings) and that these trends are (for reasons less clear to me) “analogous to those heralded in Silent Spring”. Although one sympathizes strongly with the concerns of the author, the manuscript is, in my opinion, not a serious contribution to the long overdue but badly needed policy discussion of how NIH and other medical research funding should be structured. Cynically, one might even say that the paper is yet another example of the “sky is falling in” response to any suggestion that the author’s source of support may be undergoing changes. The author surprisingly points out in the abstract that his paper was rejected by Science two-and-a-half years ago but resubmits it now with some updating; I think it still is not a substantial contribution to these policy issues.The figures and data in the paper show NIH numbers on different types of funding mechanisms and grants over the last several decades and, although the author does not explain which of these he approves of, the implication seems to be that there has been some relative decline in “the gold standard RO-1″ as calculated in various comparisons. This change apparently is apparently the cause of our current crisis. The author seems unaware that these changes, which in my analysis are really quite small, have occurred in the last decade-some initiated by the NIH leadership and some from other factors, such as congressional mandates to study child development- because of increasing skepticism about the results of the overall program. Thoughtful people and organizations have questioned whether the policies of the “golden years” are still as fruitful-whether measured by changes in public health parameters, new pharmacological agents or more subjective evaluations of our successes in dealing with the major chronic illnesses. In this era of genomics, proteomics and other aspects of “big biomedical science” the author is silent on how these needs are to be balanced with his desire for “a hundred thousand flowers”.The strange history of this paper also results in its being largely outdated at this point. Specifically, the author does not discuss the very recent creation of the National Center for the Advancement of Translational Sciences, the slightly older Centers for Translational Science Awards or the components of the ten year old NIH Roadmap (now the Common Fund). Although perhaps not in the author’s ken, these new programs have been created in response to needs articulated by other investigators. Even FASEB, which presumably supported the earlier draft of this paper, has just issued a “white paper” in support of the new NIH initiatives in translational research. I wonder if this surprises the author.I would recommend the author uses the rigor of his approach to other research problems to make his contribution to what will be a very important continued debate in the several decades ahead, especially if political and economic trends continue to limit greatly funds for medical research, about the relationship of basic and applied science, little vs big science, what planning is needed and is valuable, and many other questions.",
"responses": []
},
{
"id": "256",
"date": "05 Aug 2012",
"name": "Mark Bix",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMark Boothby’s article on the deplorable state of current NIH investigator-inititated research funding is right on target.I add my voice to the chorus of scientists witnessing and lamenting the growing struggle to maintain research funding. As this state of funding crisis continues, I believe that young talent will increasingly flee from the biomedical research enterprise. Eventually, the United States will cede to other nations the ability to generate the next bumper crop of biomedical discoveries.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-5
|
https://f1000research.com/articles/1-4/v1
|
18 Jul 12
|
{
"type": "Research Article",
"title": "Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding",
"authors": [
"Mark Livingstone",
"Michael Bidinosti",
"Michael Bidinosti"
],
"abstract": "The recent development of mammalian target of rapamycin (mTOR) kinase domain inhibitors and genetic dissection of rapamycin-sensitive and -insensitive mTOR protein complexes (mTORC1 and mTORC2) have revealed that phosphorylation of the mTOR substrate 4E-BP1 on amino acids Thr37 and/or Thr46 represents a rapamycin-insensitive activity of mTORC1. Despite numerous previous reports utilizing serine (Ser)-to-alanine (Ala) and threonine (Thr)-to-Ala phosphorylation site mutants of 4E-BP1 to assess which post-translational modification(s) directly regulate binding to eIF4E, an ambiguous understanding persists. This manuscript demonstrates that the initial, rapamycin-insensitive phosphorylation event at Thr46 is sufficient to prevent eIF4E:4E-BP1 binding. This finding is relevant, particularly as mTOR kinase domain inhibitors continue to be assessed for clinical efficacy, since it clarifies a difference between the action of these second-generation mTOR inhibitors and those of rapamycin analogues.",
"keywords": [
"The mammalian target of rapamycin (mTOR) protein is an atypical Ser/Thr protein kinase named for its well-characterized inhibition by the natural product rapamycin. Rapamycin-sensitive orthologues of mTOR exist in eukaryotes from yeast to man and are required for growth and proliferation of perhaps all eukaryotic cells. As such",
"rapamycin has been classified as an anti-fungal agent and is clinically approved as an immunosuppressant and cancer therapy1–3. Well-characterized in vivo substrates of rapamycin-sensitive mTOR activity include the 70 kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic [translation] initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The mTOR-dependent phosphorylation site on S6K1",
"Thr389",
"is required for kinase activity",
"explaining rapamycin’s inhibition of S6K1 activity4. 4E-BP1",
"on the other hand",
"is subject to multisite phosphorylation culminating in the release of bound eIF4E",
"leading to an ambiguous understanding of which phosphorylation site(s) regulate(s) eIF4E binding5."
],
"content": "Introduction\n\nThe mammalian target of rapamycin (mTOR) protein is an atypical Ser/Thr protein kinase named for its well-characterized inhibition by the natural product rapamycin. Rapamycin-sensitive orthologues of mTOR exist in eukaryotes from yeast to man and are required for growth and proliferation of perhaps all eukaryotic cells. As such, rapamycin has been classified as an anti-fungal agent and is clinically approved as an immunosuppressant and cancer therapy1–3. Well-characterized in vivo substrates of rapamycin-sensitive mTOR activity include the 70 kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic [translation] initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The mTOR-dependent phosphorylation site on S6K1, Thr389, is required for kinase activity, explaining rapamycin’s inhibition of S6K1 activity4. 4E-BP1, on the other hand, is subject to multisite phosphorylation culminating in the release of bound eIF4E, leading to an ambiguous understanding of which phosphorylation site(s) regulate(s) eIF4E binding5.\n\nMammalian 4E-BP1 is subject to an ordered phosphorylation on at least 5 major amino acid residues in response to serum-stimulation, as has been demonstrated by two-dimensional (isoelectric focusing and SDS-PAGE) electrophoresis (2DE)6. This approach of separating post-translationally modified forms of a protein based on charge and apparent molecular weight has proved to be particularly useful when combined with phosphorylation-specific antibodies7. Due to amino acid sequence similarity, phospho-specific anti-Thr37/46 antibodies did not allow determination of whether the initial phosphorylation event is at Thr37 or Thr46 using this technique, although priming phosphorylation at both of these sites is thought to be required for subsequent phosphorylation at Thr70, followed by phosphorylation at an unidentified site, and finally at Ser656,8. Given their positions flanking the amino acids responsible for eIF4E binding (amino acid residues 53–59), it is conceivable that Thr46 and Ser65 are responsible for the phosphorylation-mediated modulation of eIF4E binding occurring in response to mTOR activity. Indeed, a significant body of work supports the role of phosphorylation at Thr46 in regulating eIF4E:4E-BP1 binding9–12. Detailed analyses have led, however, to conflicting results regarding the importance of Ser65 phosphorylation in preventing this protein:protein interaction6,11,13–15.\n\nWhile rapamycin is effective in blocking phosphorylation at Thr70 and Ser65, phospho-specific antibodies to Thr37/46 show that at least one of these sites is largely rapamycin-insensitive16,17. This residual rapamycin-insensitive phosphorylation is sensitive to serum starvation, amino acid withdrawal, and non-specific phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase (PIKK) inhibitors16–19. Furthermore, the use of mTOR kinase domain inhibitors (Torin1 and PP242) in combination with mTOR complex 2 (mTORC2)-deficient cells, has allowed the determination that Thr37/46 phosphorylation represents a rapamycin-insensitive function of mTOR complex 1 (mTORC1)20,21.\n\nIn vivo studies addressing the relative importance of 4E-BP1 phosphorylation sites have been hampered by its ordered phosphorylation, wherein Thr-to-Ala mutation of Thr37 or Thr46 will block subsequent phosphorylation at Thr70 and Ser65. Mounting circumstantial evidence supports the notion that phosphorylation of Thr37/Thr46 alone is the key event regulating eIF4E:4E-BP1 binding in vivo. Intracellular co-localization of endogenous 4E-BP1 and eIF4E best correlates with dephosphorylation at Thr37/4619. 7-methyl-GTP (cap-column) pull down of eIF4E:4E-BP1 complexes is enhanced by mTOR kinase domain inhibitors more than it is by rapamycin21. Most importantly, however, mTOR active site inhibitors capable of blocking phosphorylation at Thr37/46 (and not rapamycin) induce 4E-BP-dependent phenotypes in cells22.\n\nThis manuscript describes new data demonstrating that 4E-BP1 phosphorylation at the initial, mTORC1-dependent, rapamycin-insensitive phosphorylation site is alone in regulating eIF4E binding. Furthermore, this work suggests that Thr46, and not Thr37, is this key phosphorylation site. Given the recent push for pharmaceutical development of kinase inhibitors that block both the rapamycin-sensitive and rapamycin-insensitive activities of mTOR23, a thorough understanding of the importance of rapamycin-insensitive mTORC1 activity is crucial. This manuscript supports the idea that clinically used mTOR kinase domain inhibitors will reduce eIF4E availability much more profoundly than have clinically approved rapamycin analogs.\n\n\nMaterials and methods\n\nIsoelectric focusing combined with SDS-PAGE based two-dimensional electrophoresis was performed as previously described24. Far western analyses were performed as follows using a 32P-labelled eIF4E protein25 with an N-terminal substrate peptide for heart muscle kinase (HMK). One hundred units of bovine HMK was suspended in 10 µl of 40 mM DTT and allowed to stand for 10 minutes. Five micrograms of HMK-eIF4E protein was mixed with 3 µl 10X HMK Buffer (200 mM Tris, pH 7.5, 10 mM DTT, 1 M NaCl, 120 mM MgCl2), 5ul [γ-32P] ATP 3000 Ci/mmol, 1 µl HMK (10U), and water (to 30 µl) and incubated for 45 minutes at 4°C. Probe purification was performed using Pharmacia Nick Column Sephadex G-50 DNA grade. Membranes were subjected to pre-hybridization (25 mM HEPES-KOH, pH 7.7, 25 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1% NP40, 5% skim milk) for 5 hours at 4°C. Probe hybridization was performed in buffer (20 mM HEPES-KOH, pH 7.7; 75 mM KCl, 2.5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1% NP40, 1% skim milk) with 250,000 cpm/ml of radiolabelled probe for 10 hours at 4°C. Membranes were washed with hybridization buffer 3 times, 15 minutes prior to exposure to film (BIOMAX MS, Kodak). Following far western analysis, membranes were probed sequentially with antibodies for Phospho-Ser65, Thr70, Thr37/46, and Total 4E-BP1 (Cell Signaling Technology). Cap column based fractionation of cell lysates was performed as previously described26. HeLa S3 and 293 HEK cells were treated with PP242 (2.5 µM, 30 min) or Rapamycin (10 nM, 30 min) unless otherwise indicated. Stable HeLa S3 cell lines expressing wild-type and mutant HA-4E-BP1 proteins were generated using previously described mammalian expression constructs8 and G418 selection. Control siRNA and siRNA for mTOR was from Cell Signaling Technology and delivered using Lipofectamine2000 according to manufacturer’s instructions (Invitrogen). All antibodies were from Cell Signaling Technology and were used according to manufacturer’s instructions. Compounds used were rapamycin (LC Labs), PP242 (Intellikine), torin1 (Tocris), PI-103 (EMD), etoposide and nocodazole (Sigma). Standard laboratory practices were used to control bias and unwanted sources of variability in this study. The primary limitation of the datasets presented in this manuscript is that they represent single biological replicates of an experimental procedure.\n\n\nResults\n\nFar western blot analysis, using radiolabeled HMK-eIF4E as a probe, is an effective measure of eIF4E-binding activity11,25. Using this approach, we demonstrate that the treatment of cells with the mTOR kinase inhibitor PP242 results in the following: (i) dramatic increases in the eIF4E-binding competent pool of 4E-BP1, (ii) reduction of 4E-BP1Thr37/46 and Ser65 phosphorylation, and (iii) unaffected binding to bands corresponding in molecular weight to 4E-T and eIF4G (Figure 1A). The mTOR-dependent modulation of eIF4E-binding activity is also apparent under physiological conditions (serum starvation vs. serum stimulation) and can be blocked by siRNA knockdown of mTOR in HeLa S3 cells (Figure 1B). Notably, western blot-based detection of non-phosphorylated (Thr46; denoted NP-Thr46) 4E-BP1 using a rabbit monoclonal antibody (clone: 87D12) recapitulates HMK-eIF4E binding to 4E-BP1.\n\nA) PP242 treatment increases the eIF4E binding ability of 4E-BP1 and reduces 4E-BP1 phosphorylation. B) Physiological treatment (serum starvation) modulates HMK-eIF4E binding ability of 4E-BP1 in an mTOR-dependent manner. Western blot using a non-phospho-4E-BP1/2 (Thr46) antibody parallels eIF4E binding. C) 2DE combined with far-western and western blot analysis to analyze HMK-eIF4E binding forms of 4E-BP1 under control (DMSO) and mTOR inhibitory (Rapamycin and PP242) conditions. Despite fairly equal abundance of spots A-F under control conditions, only \"spot A\" binds HMK-eIF4E. mTOR inhibition with PP242 potently increases the abundance of eIF4E-binding competent spot A, while rapamycin treatment primarily reduces phosphorylation at Ser65 (spot F).\n\nTo more precisely elucidate the molecular modifications of 4E-BP1 induced by PP242 that induce eIF4E binding, whole cell lysates were prepared from HEK293 cells that were subjected to short (30 min) treatment with PP242 or rapamycin followed by 2DE (isoelectric focusing and SDS-PAGE) and western blot analyses (Figure 1C). Under control (DMSO-treated) conditions, the previously described6 hierarchical, multi-site phosphorylation of 4E-BP1 is observed. Here, six differentially phosphorylated forms (labeled A-F) are detected by the total 4E-BP1 antibody, with forms B-F phosphorylated at Thr37 and/or Thr46, D-F phosphorylated at Thr70, and F phosphorylated at Ser65. Far western blot analysis demonstrates that only spot A is competent to bind HMK-eIF4E under control conditions suggesting that the modification responsible for spot B (Thr37 or Thr46) disrupts this interaction. Upon inhibition of mTOR with rapamycin or PP242, the predicted decrease in hyperphosphorylated 4E-BP1 forms is observed with an increase in hypophosphorylated 4E-BP1. While the identity of the phosphorylation event responsible for spot E remains undetermined, these data show that this phosphorylation site is resistant to mTOR inhibition, as rapamycin and PP242-resistant phospho-forms emanating from spot E appear above spots B-D. It is of note that the PP242-induced spot above spot B, which is not phosphorylated at Thr37 or Thr46, represents mono-phosphorylated 4E-BP1 (at the site responsible for spot E) and retains eIF4E-binding ability. The most likely candidates for this site are Thr84, which has shown to be responsible for a similarly slow SDS-PAGE migration11, and Ser101, which has been shown to promote 4E-BP1:Raptor binding27,28. The notion that Ser101 is responsible for spot E is particularly appealing, as this would provide a sound explanation for the hierarchical ordering of this phosphorylation event prior to Ser65 (spot F). That is, it is reasonable to believe that strong 4E-BP1:Raptor binding is required for complete 4E-BP1 phosphorylation, including at Ser65.\n\nNext, to determine whether Thr37 or Thr46 phosphorylation accounts for \"spot B\", which is impaired for eIF4E binding, HeLa cell lines stably expressing wild-type or mutant HA-4E-BP1 proteins were generated. Given the ordered phosphorylation of 4E-BP1, mutation of the primary phosphorylation site should block mutation of that of the subsequent phosphorylation site. For this reason, we analyzed Thr37Ala and Thr46Ala mutants in vivo using the phospho-4E-BP1 Thr37/46 antibody to determine whether the preclusion of phosphorylation at one site blocks phosphorylation at the other (Figure 2A). While endogenous 4E-BP1 was detected by the phospho-4E-BP1 Thr37/46 antibody in lysates from all stably selected cell lines, exogenous HA-tagged 4E-BP1 was poorly detected in the Thr46Ala mutant sample, suggesting that Thr46 phosphorylation is required for subsequent Thr37 phosphorylation under normal growth conditions. These results indicate that Thr46 is the initial phosphorylation site responsible for the shift from spot A to spot B, thus phosphorylation at this site alone may be sufficient to prevent 4E-BP1 binding to eIF4E (in Figure 1C). This conclusion, that Thr46 phosphorylation precedes Thr37 phosphorylation, has previously been reached by another group29.\n\nA) HeLa S3 cells stably expressing 4E-BP1 mutant proteins were subjected to western blotting using anti-HA antibody (upper), and phospho-4E-BP1 (Thr37/46) antibody (middle and lower panels). While phospho-4E-BP1 (Thr37/46) antibodies fail to detect the Thr46Ala single and Thr37/46Ala double point mutant proteins, the Thr37Ala protein is still recognized suggesting that Thr37 is not required for Thr46 phosphorylation. Endogenous 4E-BP1 phosphorylated at Thr37/46 is shown as a control. B) Untreated and Nocodazole-blocked HeLa S3 cells were subjected to cap-column pull down of eIF4E and associated proteins. eIF4E was eluted with m7-GDP followed by SDS, and fractions were analyzed by western blot using eIF4E and 4E-BP1 antibodies and phospho-4E-BP1 antibodies.\n\nThis model, wherein 4E-BP1 phosphorylation at the initial phosphorylation site (Thr46) is sufficient to prevent eIF4E binding, is also supported by 7-methyl-GTP (cap-column) pull down data (Figure 2B). Here the cap-column serves as a molecular mimic for the mRNA 5´-cap allowing eIF4E and associated binding proteins to be isolated from cell lysates. As a chemically induced pseudo-mitotic state has previously been shown to dramatically modulate the phosphorylation of 4E-BP126,30, nocodazole treatment was employed to potentially increase the diversity of 4E-BP1 phospho-forms present within our lysates. Control and nocadazole-blocked HeLa S3 cells were subjected to cap-column pull-down of eIF4E and associated 4E-BP1. This technique allowed detectable binding of only the fastest SDS-PAGE migrating forms of 4E-BP1, indicating differential binding between hypophosphorylated and hyperphosphorylated 4E-BP1 had occurred. The use of phospho-specific antibodies demonstrates that Thr37/Thr46 phosphorylated 4E-BP1 is not detectably present in this cap-column bound eIF4E fraction. Trace amounts of Thr70 phosphorylated 4E-BP1 are detected in these lanes, suggesting that mono-phosphorylated (at Thr70) 4E-BP1 exists and is eIF4E binding competent. A similar conclusion was recently reached by another group31. It should be noted that the total 4E-BP1 antibody detects a doublet band in the cap-column bound eIF4E fraction. The upper band of this doublet likely represents 4E-BP1 phosphorylated at the site responsible for the above-described \"spot E\"; a phospho-form which retains eIF4E-binding ability.\n\nTo further explore the potential existence of 4E-BP1 phospho-forms failing to adhere to the strict hierarchical phosphorylation pattern Thr37/46->Thr70->spot E->Ser65, untreated and nocodazole-blocked HeLa S3 cell lysates were mixed and subjected to 2D-E. Nocodazole block was again used to induce aberrant phosphorylation patterns of 4E-BP1. As shown in Figure 3, a pattern that was distinct from that produced under normal growth conditions emerged. Here, in addition to the normal hierarchical phosphorylation described above, alternative mono-phosphorylated species were present, as was an additional phosphorylation site causing the appearance of \"spot G\". Although, this chemical-induced atypical phosphorylation pattern may not exist under physiological conditions, these data provide support for the existence of these alternative 4E-BP1 phospho-forms.\n\nUntreated and nocodazole-blocked HeLa S3 cells lysed, pooled and subjected to 2D-E prior to analysis with phospho-specific and total 4E-BP1 antibodies (left panels). In addition to the standard ordered phosphorylation (Thr37 or Thr46, then \"spot B\", then Thr70, then \"spot E\", then Ser65, then \"spot G\"), 4E-BP1 singly phosphorylated at Thr70 is also observed indicating that this species can exist in vivo. To facilitate interpretation, the same images have been overlaid with a grid of circles corresponding to spots visible with the total 4E-BP1 antibody (right panels).\n\nRecently it has been reported that among mTOR kinase domain inhibitors, PP242 exhibits remarkably low specificity compared with Torin1, KU63794 and WYE35432. Notably, both PP242 and Torin1 were shown to bind multiple PI3K-related kinases (PIKKs), including three key DNA damage-activated kinases ATM, ATR and DNAPK, although cell-based assays failed to show inhibition of these kinases. To assess the specificity of PP242 in our system, we employed a broadly reactive phospho-specific antibody capable of recognizing multiple phospho-Ser/Thr-Gln substrates of ATM, ATR and DNAPK33,34. Indeed, with this antibody, we observed multiple etoposide-induced bands by western blot analysis (Figure 4), including bands corresponding to VCP phosphorylated at Ser784 (97 kDa) and Chk2 phosphorylated at Thr26/Ser28 (62 kDa). Remarkably, of the mTOR/PI3K inhibitors tested, only PP242 visibly reduced the etoposide-induced phosphorylation of these PIKK substrates (Figure 4A). As the concentration of PP242 (2.5 µM) was higher than that used for the other inhibitors (wortmannin 0.1 µM, PI-103 1.0 µM, Torin1 0.25 µM and Rapamycin 0.01 µM), PP242 pretreatment was also assessed at 0.34, 0.68 and 1.25 µM, and gave similar results (Figure 4B).\n\nA) Western blot analysis of lysates from HeLa cells pretreated with the indicated inhibitors prior to etoposide treatment reveals that PP242 reduces the appearance of multiple phospho-SQ/TQ epitopes recognized by the broadly reactive phospho-VCP/Chk2 antibody. B) Varying concentrations of PP242 from 0.34–1.25 µM block etoposide-induced phosphorylation of proteins recognized by the phospho-SQ/TQ antibody.\n\n\nDiscussion\n\nAlthough, our conclusion that phosphorylation at Thr46 is the key event regulating 4E-BP1:eIF4E binding has been suggested previously, none of these previous studies unambiguously established that Thr46 alone is the key important site. These studies either: (i) employed in vivo phosphorylation of a Thr46Ala point mutant also blocking subsequent phosphorylation events9,11; (ii) failed to identify a single important phosphorylation site10,11; or (iii) employed in vitro phosphorylation of Thr46Ala point mutant using a non-physiological kinase11,12.\n\nImportantly, equally credible work has reported that phosphorylation on Thr46 is unimportant in the regulation of 4E-BP1:eIF4E binding8,35. It is interesting to note that both of these studies evaluate the capacity of in vitro phosphorylation at Thr46 to disrupt pre-existing 4E-BP1:eIF4E complexes and utilize cap column purification to isolate eIF4E-bound 4E-BP1. This experimental detail is particularly relevant, as it has been shown that the RNA cap can exert an allosteric effect stabilizing 4E-BP1:eIF4E binding14,36. Taken together, our data and these previous reports could suggest that Thr46 phosphorylation is sufficient to block the initial binding between eIF4E and 4E-BP1 as observed by far western analyses, but that hyperphosphorylation of 4E-BP1, including at Ser65, is required to disrupt existing 4E-BP1:eIF4E complexes.\n\nThe far western analysis of 2DE separated 4E-BP1 phospho-forms presented in Figure 1C is apparently at the limit of its useful range of detection. While this technique allows a comparison of binding efficiencies of spots A and B to evaluate the impact of Thr46 phosphorylation, it is not useful to compare the relative binding abilities of spots E and F to allow a similar assessment of the importance of Ser65 phosphorylation. Perhaps with a more sensitive assay we would have observed a similar decrease in eIF4E binding upon Ser65 phosphorylation. This would be evidence that Thr46 phosphorylation is sufficient to block the initial binding of 4E-BP1 to eIF4E when both proteins are present at low physiological concentrations, but that phosphorylation at multiple sites culminating at Ser65 is required to prevent/disrupt binding when the two proteins are present at high concentrations/local concentrations.\n\nThe data presented in Figure 2B at first seem to be at odds with this theory that 4E-BP1 phosphorylated at Thr37 or Thr46 could be pre-associated with mRNA 5´ cap-bound eIF4E in cells. This technique assesses, however, the de novo association of eIF4E (4E-BP1 associated or not) with an mRNA 5´ cap analog, therefore any pre-existing complexes must dissociate from cellular mRNA caps prior to isolation. These data do tell us that if there is some pool of 4E-BP1 phosphorylated on Thr46 associated with eIF4E, then this complex is not able to efficiently bind the mRNA cap analog. This assay may also evaluate the sequential binding of eIF4E to the cap followed by 4E-BP1 binding to cap-associated eIF4E and indicate in agreement with the far western data in Figure 1C that de novo binding is blocked by Thr46 phosphorylation.\n\nFigure 2B and Figure 3 demonstrate that blocking cells in a pseudo-mitotic state with nocodazole results in phosphorylation of 4E-BP1 at up to 6 distinct phosphorylation sites, a finding at odds with previous work demonstrating hypophosphorylation of 4E-BP1 in mitosis26. The polyclonal antibody, referred to as 11208 and used in this previous study, however, exhibits selectivity for the non-phosphorylated forms of the protein after nocodazole treatment (ML, unpublished). A similar faulty antibody-based discrepancy regarding the phosphorylation state of 4E-BP1 in meiotic oocytes was resolved37. While we observed at most 6 different phospho-forms of 4E-BP1, the PhosphoSitePlus database38 indicates that 20 distinct phosphorylation sites have been described in the literature and/or mass spectrometry-based datasets. This discrepancy suggests that many of these 20 phosphorylation events are either mutually exclusive, or occur only rarely, not at all, in response to specific stimuli, or in specific cell types.\n\nFinally, the demonstration presented within this manuscript that PP242 exhibits poor specificity for mTOR in a cell-based assay for PIKK off-target effects likely does not change the conclusions of this manuscript; however, it serves as a reminder that any new mTOR inhibitor may have unanticipated effects. While the inhibition of mTORC2 by mTOR kinase domain inhibitors is expected to have profound effects, the evidence presented in this manuscript suggests that the rapamycin-insensitive activity of mTORC1 towards 4E-BP1 will also be quite important as the clinical safety and efficacy of mTOR kinase domain inhibitors is assessed.",
"appendix": "Author contributions\n\n\n\nML conceived the study, designed the experiments, and prepared the first draft of the manuscript. ML and MB carried out the research, were involved in the revision of the draft manuscript, and have approved the final content.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests declared.\n\n\nGrant information\n\nML was a research student of the Terry Fox Foundation (Award #700029) and is currently supported by the Pasteur Foundation.\n\n\nAcknowledgements\n\nWe thank Marc R. Fabian for assistance with far western analyses.\n\n\nReferences\n\nVezina C, Kudelski A, Sehgal SN: Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomyceteand isolation of the active principle. J Antibiot (Tokyo). 1975; 28(10): 721–726. PubMed Abstract | Publisher Full Text\n\nMartel RR, Klicius J, Galet S: Inhibition of the immune response by rapamycin, a new antifungal antibiotic. Can J Physiol Pharmacol. 1977; 55(1): 48–51. PubMed Abstract\n\nHudes GR, Berkenblit A, Feingold J, et al.: Clinical trial experience with temsirolimus in patients with advanced renal cell carcinoma. Semin Oncol. 2009; 36(Suppl 3): S26–36. PubMed Abstract | Publisher Full Text\n\nPearson RB, Dennis PB, Han JW, et al.: The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J. 1995; 14(21): 5279–5287. PubMed Abstract | Free Full Text\n\nBeretta L, Gingras AC, Svitkin YV, et al.: Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J. 1996; 15(3): 658–664. PubMed Abstract | Free Full Text\n\nGingras AC, Raught B, Gygi SP, et al.: Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 2001; 15(21): 2852–2864. PubMed Abstract | Free Full Text\n\nKovarova H, Hajduch M, Livingstone M, et al.: Analysis of state-specific phosphorylation of proteins by two-dimensional gel electrophoresis approach. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 787(1): 53–61. PubMed Abstract | Publisher Full Text\n\nGingras AC, Gygi SP, Raught B, et al.: Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev. 1999; 13(11): 1422–1437. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurnett PE, Barrow RK, Cohen NA, et al.: RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci USA. 1998; 95(4): 1432–1437. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFadden P, Haystead TA, Lawrence JC Jr: Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes. J Biol Chem. 1997; 272(15): 10240–10247. PubMed Abstract | Publisher Full Text\n\nMothe-Satney I, Yang D, Fadden P, et al.: Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression. Mol Cell Biol. 2000; 20(10): 3558–3567. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang D, Brunn GJ, Lawrence JC Jr: Mutational analysis of sites in the translational regulator, PHAS-I, that are selectively phosphorylated by mTOR. FEBS Lett. 1999; 453(3): 387–390. PubMed Abstract | Publisher Full Text\n\nTomoo K, Abiko F, Miyagawa H, et al.: Effect of N-terminal region of eIF4E and Ser65-phosphorylation of 4E-BP1 on interaction between eIF4E and 4E-BP1 fragment peptide. J Biochem. 2006; 140(2): 237–246. PubMed Abstract | Publisher Full Text\n\nTomoo K, Matsushita Y, Fujisaki H, et al.: Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods. Biochim Biophys Acta. 2005; 1753(2): 191–208. PubMed Abstract | Publisher Full Text\n\nTait S, Dutta K, Cowburn D, et al.: Local control of a disorder-order transition in 4E-BP1 underpins regulation of translation via eIF4E. Proc Natl Acad Sci USA. 2010; 107(41): 17627–17632. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFan QW, Knight ZA, Goldenberg DD, et al.: A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell. 2006; 9(5): 341–349. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang X, Beugnet A, Murakami M, et al.: Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin factor 4E-binding proteins. Mol Cell Biol. 2005; 25(7): 2558–2572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRong L, Livingstone M, Sukarieh R, et al.: Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs. RNA. 2008; 14(7): 1318–27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLivingstone M, Larsson O, Sukarieh R, et al.: A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E. Chem Biol. 2009; 16(12): 1240–1249. PubMed Abstract | Publisher Full Text\n\nFeldman ME, Apsel B, Uotila A, et al.: Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2. PLoS Biol. 2009; 7(2): e38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThoreen CC, Kang SA, Chang JW, et al.: An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem. 2009; 284(12): 8023–8032. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDowling RJ, Topisirovic I, Alain T, et al.: mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science. 2010; 328(5982): 1172–1176. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBenjamin D, Colombi M, Moroni C, et al.: Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat Rev Drug Discov. 2011; 10(11): 868–880. PubMed Abstract | Publisher Full Text\n\nBidinosti M, Ran I, Sanchez-Carbente MR, et al.: Postnatal deamidation of 4E-BP2 in brain enhances its association with raptor and alters kinetics of excitatory synaptic transmission. Mol Cell. 2010; 37(6): 797–808. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPause A, Belsham GJ, Gingras AC, et al.: Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5´-cap function. Nature. 1994; 371(6500): 762–767. PubMed Abstract | Publisher Full Text\n\nPyronnet S, Dostie J, Sonenberg N: Suppression of cap-dependent translation in mitosis. Genes Dev. 2001; 15(16): 2083–2093. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeugnet A, Wang X, Proud CG: Target of rapamycin (TOR)-signaling and RAIP motifs play distinct roles in the mammalian TOR-dependent phosphorylation of initiation factor 4E-binding protein 1. J Biol Chem. 2003; 278(42): 40717–40722. PubMed Abstract | Publisher Full Text\n\nWang X, Li W, Parra JL, et al.: The C terminus of initiation factor 4E-binding protein 1 contains multiple regulatory features that influence its function and phosphorylation. Mol Cell Biol. 2003; 23(5): 1546–1557. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHerbert TP, Tee AR, Proud CG: The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. J Biol Chem. 2002; 277(13): 11591–11596. PubMed Abstract | Publisher Full Text\n\nHeesom KJ, Gampel A, Mellor H, et al.: Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1). Curr Biol. 2001; 11(17): 1374–1379. PubMed Abstract | Publisher Full Text\n\nAyuso MI, Hernandez-Jimenez M, Martin ME, et al.: New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress. J Biol Chem. 2010; 285(45): 34355–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu Q, Kirubakaran S, Hur W, et al.: Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics. J Biol Chem. 2012; 287(13): 9742–9752. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLivingstone M, Ruan H, Weiner J, et al.: Valosin-containing protein phosphorylation at Ser784 in response to DNA damage. Cancer Res. 2005; 65(17): 7533–7540. PubMed Abstract\n\nStokes MP, Rush J, Macneill J, et al.: Profiling of UV-induced ATM/ATR signaling pathways. Proc Natl Acad Sci USA. 2007; 104(50): 19855–19860. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeesom KJ, Denton RM: Dissociation of the eukaryotic initiation factor-4E/4E-BP1 complex involves phosphorylation of 4E-BP1 by an mTOR-associated kinase. FEBS Lett. 1999; 457(3): 489–493. PubMed Abstract | Publisher Full Text\n\nShen X, Tomoo K, Uchiyama S, et al.: Structural and thermodynamic behavior of eukaryotic initiation factor 4E in supramolecular formation with 4E-binding protein 1 and mRNA cap analogue, studied by spectroscopic methods. Chem Pharm Bull (Tokyo). 2001; 49(10): 1299–1303. PubMed Abstract | Publisher Full Text\n\nEllederova Z, Kovarova H, Melo-Sterza F, et al.: Supression of translation during in vitro maturation of pig oocytes despite enhanced formation of cap-binding protein complex eIF4F and 4E-BP1 hyperphosphorylation. Mol Reprod Dev. 2006; 73(1): 68–76. PubMed Abstract | Publisher Full Text\n\nHornbeck PV, Kornhauser JM, Tkachev S, et al.: PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 2012; 40(Database issue): D261–270. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "250",
"date": "23 Jul 2012",
"name": "David Fruman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThrough binding to eIF4E, the 4EBP proteins (4EBP1, 2, 3) interfere with formation of the eIF4F cap-binding complex. A key function of the mTOR serine-threonine kinase is to phosphorylate 4EBPs on multiple sites, which blocks their ability to bind to eIF4E thus potentiating cap-dependent translation.Recently it has become clear that ATP-competitive mTOR kinase inhibitors are more effective than the allosteric compound rapamycin at blocking mTOR-dependent 4EBP phosphorylation, particularly on sites T37 and T46. This greater effect of mTOR kinase inhibitors on 4EBP phosphorylation correlates with increased anti-proliferative and pro-apoptotic effects. The purpose of the current study is to determine the order of phosphorylation of sites T37 and T46, and which site is most important for regulating binding of 4EBP1 to eIF4E. This is a fairly narrow question to address but the authors argue that it is important for understanding drug mechanism and that previous approaches have not addressed this question adequately.Overall the data support the conclusion that T46 phosphorylation is the initial, rapamycin-insensitive event that blocks 4EBP1-eIF4E binding. The main approach used is two-dimensional electrophoresis (IEF followed by SDS-PAGE), then far-western blotting with labeled eIF4E to determine which spots are capable of eIF4E binding. Figure 1A provides a 1-D blot that shows convincingly that the far-western technique can reveal increased binding of eIF4E to 4EBP1 in cells treated with the mTOR kinase inhibitor PP242. Figure 1B shows that 4EBP1 phosphorylation and release from eIF4E are induced by serum in a mTOR-dependent manner. Figure 1C presents the 2D data illustrating hierarchical phosphorylation and distinct effects of rapamycin and PP242. The data support the conclusion that only a small subset of 4EBP forms are capable of binding eIF4E, that these forms lack T37/46 phosphorylation, and that PP242 increases this pool to a greater extent than rapamycin. To distinguish the roles of T37 and T46 they express HA-tagged 4EBP1 and alanine substitution mutants in HeLa cells. The data in Figure 2A show that T46A mutation, but not T37A mutation, blocks detection by the phospho-T37/46 antibody. This suggests that T46 phosphorylation precedes and is required for T37 phosphorylation. A cap binding assay in Figure 2B supports the conclusion that T37/46-phosphorylated 4EBP1 cannot bind eIF4E in the cap-binding complex, but this is not novel and the technique does not add further evidence for initial T46 phosphorylation.The rest of the data seem added on and superfluous to the main message of the paper. The experiment in Figure 3 shows 2D far western blotting data on lysates of nocodazole-treated cells, which artificially increases the diversity of 4EBP1 spots. As the authors point out, these might not exist under physiological conditions so the relevance is unclear. Figure 4 addresses an entirely different problem, namely the lack of selectivity of the PP242 compound. While it is of some interest that PP242 differs from other compounds of this class in its apparent inhibition of DNA-PK and related kinases, this observation is too peripheral to the main study and should be developed further before publication.",
"responses": []
},
{
"id": "252",
"date": "30 Jul 2012",
"name": "Christopher G. Proud",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "253",
"date": "02 Aug 2012",
"name": "Olaf Isken",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis submission is quite nice for experts, but not particularly well-written for those outside of the field of 4E-BP1 phosphorylation, who will have difficulty reading it ; hence my suggested clarifications below. The article is important as it demonstrates that the use of mTOR-kinase domain inhibitors would be more efficacious than the use of rapamycin analogs as therapeutics.The work clarifies that phosphorylation of 4E-BP1 at Ser65 by mTOR is not required to inhibit 4E-BP1 binding to eIF4E and that rapamycin-insensitive mTOR-mediated phosphorylation at 4E-BP1 Thr46 is sufficient to inhibit 4E-BP1 binding to eIF4E. Phosphorylation at 4E-BP1 Thr37 is also shown to be rapamycin-insensitive. Notably, evidence that phosphorylation at Thr 37 or Thr 46 appears to be required for phosphorylation at Thr 70 and Ser 65 supported the notion that the earlier phosphorylation events were key to promoting translation.Improvement Suggestions:Page 2, first paragraph, last sentence. The meaning of “4E-BP-dependent phenotypes” is not particularly clear.Page 2, line four of Materials and Methods. Shouldn’t “32″ be superscript?Page 2, last paragraph. The statement “Here, six differentially phosphorylated forms (A-F) …” is confusing. Is form A really phosphorylated? Earlier in the manuscript, the authors mentioned 5 major phosphorylation sites.In Figure 1, the assay used in this Figure requires much better description in the text. Part A of the legend requires specification of the experimental approach. Also, the title of Figure 1 could be more useful if it were informative/conclusive rather than descriptive.Page 3, right column, second paragraph. The sentence “While endogenous… , suggesting that Thr46 phosphorylation is required for subsequent The37 phosphorylation under normal growth conditions.” is misleading since 4E-BP1 mutant (Thr46Ala) is clearly phosphorylated at Thr37 in Fig. 2A middle panel. With the data presented here the reader is left wondering whether the differences in detecting HA-4E-BP1 (T37A) or HA-4E-BP1 (T46A) might be simply due to different affinities of the (The37/46) antibody used.What is the difference between the middle and the lower panel? Are the antibodies used to probe the middle and the lower panel (e.g. phosho-HA-4E-BP1 (Thr37/46) vs. phosho-4E-BP1 (Thr37/46)) different or the same? When using cell lysates of stable cell lines and a phospho-4E-BP1 (Thr37/46) antibody, shouldn’t be HA 4E-BP1 wildtype and mutant forms be detectable in the lower panel as well?Page 3, last paragraph, line 8. “modulate” would be clearer if described as the specific modulations that occur – in the literature a state of 4E-BP1 hypophosphorylation. The author understands that the changes are complicated and clarified from these authors’ perspective in Figure 3 and in the Discussion. However, possibly, the reader could be better informed at this point of the manuscript.Page 4, second paragraph, right-hand column. Please clarify the effects and usefulness of etoposide and the meaning of VCP and, in the legend, VCP/Chk2.In Figure 2B, the authors should provide a control western blot of an unrelated protein which does not belong to the eIF4E-associated proteins under normal conditions to show the unspecific background of this assay.It is not clear to this reader why mono-phosphorylated 4E-BP1 (at position Thr70) is detectable in the non-Nocadazole treated cell samples in Fig. 2B (lane 3 and 5) when in Fig.3 they show that mono-phosphorylation of 4E-BP1 at position Thr70 is triggered by a Nocodazol-induced block of Hela cells but not detectable under normal conditions.In Figure 3, why did the authors mix Control and Nocodazole-blocked Hela cell lysates instead of showing them side-by-side? Also, why are there additional spots in the interpretation panels for Phos-Thr70 and Phos-Thr37/46 that have no counterpart in the respective western blot panels? This figure is already difficult, so the authors should be careful with the overlays.In Figure 4, all results in this Figure are not adequately described. For example, the authors need to explain the analysis of ubiquitin and its significance to their studies.The sentence “ …rapamycin-insensitive phosphorylation site is alone in regulating eIF4E binding.” should read something like “ …rapamycin-insensitive phosphorylation site is alone responsible in regulating eIF4E binding”.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-4
|
https://f1000research.com/articles/1-3/v1
|
16 Jul 12
|
{
"type": "Research Article",
"title": "Low budget analysis of Direct-To-Consumer genomic testing familial data",
"authors": [
"Gustavo Glusman",
"Mike Cariaso",
"Rafael Jimenez",
"Daniel Swan",
"Bastian Greshake",
"Jong Bhak",
"Darren W Logan",
"Manuel Corpas",
"Gustavo Glusman",
"Mike Cariaso",
"Rafael Jimenez",
"Daniel Swan",
"Bastian Greshake",
"Jong Bhak",
"Darren W Logan"
],
"abstract": "Direct-to-consumer (DTC) genetic testing is a recent commercial endeavor that allows the general public to access personal genomic data. The growing availability of personal genomic data has in turn stimulated the development of non-commercial tools for DTC data analysis. Despite this new wealth of public resources, no systematic research has been carried out to assess these tools for interpretation of DTC data. Here, we provide an initial analysis benchmark in the context of a whole family, using single nucleotide polymorphism (SNP) data. Five blood-related DTC SNP chip data tests were analyzed in conjunction with one whole exome sequence. We report findings related to genomic similarity between individuals, genetic risks and an overall assessment of data quality; thus providing an evaluation of the current potential of public domain analysis tools for personal genomics. We envisage that as the use of personal genome tests spreads to the general population, publicly available tools will have a more prominent role in the interpretation of genomic data in the context of health risks and ancestry.",
"keywords": [
"Direct-to-Consumer (DTC) genetic testing is a relatively new commercial endeavor offering access to personal genomic tests to the general public. Individuals wishing to learn about their genomes today enjoy a range of options. DTC providers typically offer chip-based genotyping of genome-wide markers",
"currently in the range of hundreds of thousands to a million single-nucleotide polymorphisms (SNPs). This current wealth of personal genomic data is likely to grow at an increasing pace as DNA sequencing become ubiquitous in personal genome testing. Genome sequencing allows elucidation of not just SNPs",
"but copy number variants (CNVs)",
"insertions",
"inversions and many other genomic features currently underrepresented in personal genome analyses. Yet personal SNP data has proven to be a valuable resource for making personalized inferences about the risk of developing medical conditions",
"the probability of having certain phenotypic traits",
"and one’s likely ancestral origins1",
"2. Taking advantage of the growing body of statistical associations accumulated in the scientific literature",
"DTC providers have been able to offer personalized genomic ‘reports’ that present accessible scientific information of relevance to their customers’ observed genotypes. It is precisely in these genomic annotations where customers realize the value of their DTC product purchase."
],
"content": "Introduction\n\nDirect-to-Consumer (DTC) genetic testing is a relatively new commercial endeavor offering access to personal genomic tests to the general public. Individuals wishing to learn about their genomes today enjoy a range of options. DTC providers typically offer chip-based genotyping of genome-wide markers, currently in the range of hundreds of thousands to a million single-nucleotide polymorphisms (SNPs). This current wealth of personal genomic data is likely to grow at an increasing pace as DNA sequencing become ubiquitous in personal genome testing. Genome sequencing allows elucidation of not just SNPs, but copy number variants (CNVs), insertions, inversions and many other genomic features currently underrepresented in personal genome analyses. Yet personal SNP data has proven to be a valuable resource for making personalized inferences about the risk of developing medical conditions, the probability of having certain phenotypic traits, and one’s likely ancestral origins1,2. Taking advantage of the growing body of statistical associations accumulated in the scientific literature, DTC providers have been able to offer personalized genomic ‘reports’ that present accessible scientific information of relevance to their customers’ observed genotypes. It is precisely in these genomic annotations where customers realize the value of their DTC product purchase.\n\nA feature of DTC genomic test interpretation is that, being a commercial product, genomic annotations and analysis tools are proprietary and not freely available to the research community. This has motivated the parallel development of public resources and low cost genotype analysis tools. SNPedia3 is a wiki-styled resource that collects and annotates SNPs from the scientific literature and provides tools with which to associate these annotations to those observed in DTC genomic tests. openSNP4 is a public resource that collects genotypes from people willing to share them, allows annotation of phenotypes, and the search of occurrences of a particular SNP in scientific publications using Mendeley5. Although all resources, public or commercial, are limited by the reliability of the data available for any given marker, public and low cost resources have the potential of engaging community wide efforts (‘crowd-sourcing’) to an extent to which closed commercial applications cannot.\n\nWe decided to explore the extent to which phenotype inference and genotype analysis can be carried out solely using existing public or very low cost resources. This is motivated by our belief that no DTC company will ultimately be able to match the rapid pace of genomic data accumulation and annotation that the research community is producing. Apart from SNPedia or openSNP, a wiki-based model, perhaps integrated with existing genetics resources such as the Gene Wiki6 or Gene Wiki+7, may offer a good solution for rapid, accurate and comprehensive community annotation of personal genomic data.\n\nIn this paper we carry out a systematic analysis of DTC genomic data from a family of five blood relatives using mostly public annotations and tools. We present a) our findings related to the quality of the data, b) a comparison of the similarity between members of the family and an undisclosed individual of a different ethnic background and c) phenotype inferences as described by SNPedia trait annotations. We also incorporate analysis of DTC exome sequence data to supplement the genotype findings of one individual. We thus offer a pioneering methodical study of a whole family analyzed using only DTC data. Since comparable data could, in principle, be bought by any individual, this study benchmarks the personal genomic analyses available to non-experts using open, web-based tools.\n\n\nResults\n\nWe analyzed DTC genomic data from a family of five of self-reported European ancestry, two males and three females across two generations (Figure 1).\n\nSquares and circles denote male and female respectively. Filled shapes represent those for which genome data is available. ‘Son’, the individual whose exome was sequenced, is denoted with a red diamond. Other family members include Father, Mother, Daughter and Aunt, who is Mother’s sister.\n\nThe family has lived in the southern-most region of Western Europe (Andalusia, Spain) for at least 4 generations. Principal component analyses of ancestry informative markers confirm tight parental clustering with Southern European populations (Figure 2). We thus expect their ethnic background to be relatively homogeneous. The CEU HapMap ethnic group8 was taken as the reference genotype for SNPedia phenotype predictions. Two kinds of SNP chip were used in this analysis, the 23andMe9 versions 2 and 3. All family members except ‘Son’ (denoted with red diamond in Figure 1) were tested with version 3. Son was tested using version 2 and whole exome sequencing.\n\nMother (ES7) is denoted by a red arrow and Father (ES8) by a blue arrow. Mother and Father are the only family individuals included here as they have the most divergent genotypes within the family.\n\nWhole family genotypes provide an additional genetic context that individual data analyses cannot offer, enabling enhanced error correction and inheritance state analysis10. We found that data downloaded from 23andMe at different times may vary, probably as a consequence of changes in genotype-calling algorithms. To ensure consistency in our analyses, we downloaded the most recent data, for all family members, on May 30th 2012.\n\n\n\nIn order to facilitate comparison between different genotypes, we excluded non-autosomal SNP data (chromosomes X, Y and MT). All v3 chips had a total of 930,342 autosomal SNPs; the v2 SNP chip had 556,694. The reported ‘no call’ rate (shown as ‘- -’ in the downloaded data) varied slightly for each individual (Table 1), but overall, when genotypes are expressed as percentages of the total, some differences are observed for Son (v2) when compared to all other v3 individuals (Figure 3). V3 individuals show a very similar distribution of genotypes.\n\nFor the purposes of unbiased comparison, only autosome data is included. I and D indicate insertion and deletion, respectively. Son’s percentages (v2) show slight differences to all other v3 individuals whose genotype proportions are more similar.\n\n23andMe reports a 98% or greater call rate11, meaning that the chip can provide accurate data for more than 98% of those variants in any particular person. When an allele variant present in heterozygous state is \"undercalled\" (not observed), the locus may be reported as being homozygous for the other variant, leading to missed heterozygosity. Such sites may significantly impact the disease risks predicted for the individual. Under the simplifying assumption of uniform undercall probability, we estimated the number of heterozygous sites mistakenly reported as homozygous (Table 2). This means that for Son, 1 in every 400 sites is mistakenly called. For Father, 1 in every 200. Next, we analyzed Mendelian Inheritance Errors (MIEs). If at one site a reported genotype is ‘CC’ but the genotypes for both parents is ‘TT’, one possible explanation is that one of the parents is actually heterozygous ‘CT’ but was undercalled as ‘CC’, and likewise the son is heterozygous ‘CT’ but was undercalled ‘TT’. Given 5 people, there are 10 possible pairwise relations. Four of these represent direct parent/offspring relations, for which discrepancies can be counted as MIEs (Table 3).\n\nOne SNP in the Daughter (chr4:7, 9957, 622) is in disagreement with both parents: Father=CC, Mother=CC, Daughter=TT.\n\nFor the remaining 6 relationships, only part of the genome is expected to be identical by descent (IBD). Fully incompatible sites can be numbered as \"pseudo-MIEs\" (Table 4).\n\nThe reduced numbers of MIEs and pseudo-MIEs between the Son and all other family members were due to the lower total number of SNPs assayed for the Son. The Daughter has more MIEs relative to the Mother than relative to the Father. This may be due to having inherited a few deleted segments leading to a hemizygous state: hemizygous sites are reported as homozygous for the allele present, leading to an accumulation of apparent MIE sites. For example, at chr2:41093584 (rs12465519) (Table 5). The ISCA analysis explains how we inferred a deletion from observed discordant genotypes.\n\n\n\nWe compared the 23andMe genotypes of the family members at three levels: 1) pairwise all-against-all comparison, 2) inheritance state analysis in family quartets, and 3) analysis of population admixture.\n\n1. Pairwise comparison. We performed an all-against-all genotype comparison among all family members. As an external point of reference, we included in our analysis a male individual of Indian ethnic background (denoted as non-CEU, chip version 3). Table 6 shows a summary of the SNP similarity patterns found among all family members and the non-CEU individual. When comparing the similarity of tested SNPs between family members (for information on how similarity scores are calculated see Methods section) we find that Son is most similar to Daughter (85.7%), then to Father (83.8%) and Mother (83.7%), and least to Aunt (78.7%) and the non-CEU individual (75.4%). Among individuals sharing the same platform, we find that siblings, Aunt and Mother share the greatest number of identical genotypes (84.5%). When comparing whose SNPs are the most similar to Father, neither Mother nor Aunt are found significantly different (p-value = 0.5561). The non-CEU individual has, on average, a similarity of 75.3% to all family members tested on platform v3. Contrary to our expectation that both Sister and Son should be equally similar to Mother and Father, we found that Daughter is 84.8% identical to Mother and 85.0% to Father, potentially explained by the inheritance of a deletion (see previous section). This difference Father/Daughter vs. Mather/Daughter similarity was found significant (p-value = 1.731e-05). Son however, exhibited 83.7% identity to Mother and 83.8% to Father and his tested SNPs were not found to be significantly different to either of them (p-value = 0.2751).\n\nMatches denote identical genotypes for the same SNP (e.g. AA/AA); half-match, only one of the alleles is identical (e.g. AT/AA) and conflict means both alleles are different (e.g. CG/AT).\n\n2. Inheritance state analysis. The availability of nuclear families with two or more offspring enables the identification of inheritance states10. These represent whether the offspring inherited the same alleles from both parents (\"identical\" state), the same allele from one parent but different alleles from the other (\"haploidentical\" state, maternal or paternal according to the parent from which the same allele was inherited), or different alleles from both parents (\"nonidentical\" state). Some family genotypes are consistent with all inheritance states (e.g. when all family members are homozygous) and are thus non-informative. Some family genotypes are consistent only with a subset of the inheritance states. For example if both parents are heterozygous A/G, and both offspring are homozygous A/A, clearly the offspring inherited the same alleles from both parents, which is consistent only with the \"identical\" state. Some family genotypes are consistent with two inheritance states. By combining the evidence from individual SNPs along a chromosome, it is possible to identify contiguous blocks of consistent inheritance, bounded by recombination events in either parent (Figure 3). The overall fraction of the genome present in each inheritance state (Figure 3 inset) deviates little from the expected 25%.\n\nWe found that 23andMe genotypes are sufficient for performing this analysis and identifying well-defined inheritance state blocks, despite covering a very small fraction of the genome (approx. half a million shared SNPs, limited by the lower density version used for Son). This is due to the largely uniform sampling of SNPs along the genome.\n\n3. Admixture analysis. Visualization of admixture for Mother and Father was done with ADMIXTURE12 in the context of other similar Southern European individuals. Figure 2 showed an admixture mapping for a selection of individuals from the Eurogenes Genetic Ancestry project13. Mother was denoted as ES7 (red arrow) and Father as ES8 (blue arrow). Mother and Father seemed to be markedly different yet in and around the Portuguese and Spanish cluster of individuals.\n\nTo leverage all genotype data contained in all different sources, we combined the SNP chip data v2 (574,406 SNPs) with those found in Son’s exome data. 10,203 genotypes were annotated in SNPedia when exome SNPs were pooled with the SNP chip version 2 (generated on 11th June, 2012). Processing just the exome, only 925 genotypes were found annotated in SNPedia. It is not surprising that so few additional SNPs were found, as the exome comprises a very small percentage of the total genome. Two SNPs were discovered to have conflicting genotypes between the two platforms: rs12344615 reported as ‘AG’ and ‘GG’ and rs2290272 reported as ‘CT’ and ‘TT’ respectively. The most likely informative SNPs from the exome data are summarized as judged by their observed frequency in HapMap8.\n\n\n\nFor the analysis of similarities between genotypes only 23andMe data was analyzed. Although exome sequencing is not widely marketed yet within the DTC providers as an option, this is likely to change in the near future. With our current budget constraints, we were able to sequence Son’s exome and relevant SNP and variation data was added for further analysis. A BAM file was created out of 4 FASTQ files downloaded from a server from the Beijing Genomics Institute. A compressed VCF file was also created. A total of 2.54 Gigabases of sequence was aligned at high quality. Summary metrics of the exome were calculated using Picard14 and showed a minimum of 61.42% of the on-target regions were covered with a depth at least 20x. Genotyping with GATK15 identified 37,702 variations relative to the reference genome (GRCh37). This was noted to be lower than expected if additional samples had been genotyped concurrently. 97% of the variants identified were within a known gene, 58% of them overlapped a protein domain and 5,565 were non-synonymous (15%) with serious predicted consequences on the protein product (as determined by SIFT16, PolyPhen17 or Condel18). Of these 5,565 potentially pathogenic SNPs, 413 had not been previously identified (verified against dbSNP release 132). This represents a normal figure for the private, novel, non-synonymous changes carried by most individuals. No more serious, novel changes were identified (such as stop codons gained or lost).\n\nWe used SNPedia’s Hilbert curve visualization tool to compare chromosomes between different individuals. Figure 5 shows an example of a Hilbert curve comparison between chromosome 1 of Mother and all family members and the non-CEU individual. Each pixel corresponds to a SNP, colored according to four categories: match (light blue), half-match (dark blue), mismatch (red) and no data (grey). Two patches of light blue are apparent in the Mother-Aunt comparison of chromosome 1, and more appear in other chromosomes (not shown). These correspond to ‘identical’ segments in which the Mother and the Aunt inherited identical haplotypes from both their parents.\n\n\n\nSNPs consistent with two inheritance states contribute 0.5 weight to each. SNP counts are binned in non-overlapping 1 Mb windows; within each window, the four inheritance states are sorted by decreasing level of support. Regions without support typically overlap centromeric repeats and heterochromatic regions. Pie chart inset: fraction of the genome observed in each inheritance state.\n\nEach pixel represents a SNP. Light blue represents match, dark blue half-match and red conflict. SNPs in Son that are not present in the genotypes of the other individuals are represented in grey.\n\nThe next most similar graph can be seen to be the Mother/Daughter comparison. Much of the graph comparing Mother and Son is grey, representing SNPs present only on 23andMe v3 but absent from v2.\n\n\n\nWe inferred phenotypes from the observed genotypes, by comparing to all available SNPedia SNP annotations. We analyzed family genotypes using the Promethease tool19, which allows annotation of observed genotypes from DTC analyses with SNPedia-annotated phenotype associations. These are collected directly from the scientific literature. All family members were analyzed using Promethease and associated phenotypes were collected for further interpretation. SNPedia’s annotation Magnitude, denoting a subjective degree of importance for an observed trait as judged by the curator, was used to discriminate between traits that should be considered further in our analyses. A magnitude of 0 denotes a common genotype for which no associated phenotypic data is known. Magnitude >3 is defined as ‘probably’ interesting. The maximum number is 10. In order to compare family phenotype annotations, all SNPs or ‘genosets’ (groups of SNPs) with equal or greater than magnitude 3 were extracted and summarized in Table 5. Results for Son are not directly comparable as he has fewer SNPs analyzed.\n\nTraits have been classified according to the general condition they relate. Red boxes are indicative of a particular phenotype being predicted in the individual. Descriptions for every matched phenotype, extracted directly from SNPedia, are shown in the right column.\n\nBased on observed results, the maternal hereditary line seems to carry greater risks related to diabetes and cardiovascular/thrombosis related conditions. In addition, both males (Son and Father) have greater risk of baldness as well as mixed results in terms of their ability to metabolize drugs. All the family shares a common trait of substantially increased odds of developing V617F-positive Myeloproliferative neoplasms.\n\n\nDiscussion\n\nWe have presented here, to our knowledge, the first systematic analysis of DTC genomic data using non-commercial or low cost resources, combining data from 5 blood-related family members. The purpose of this study does not lie in uncovering the phenotypic predictions or genetic findings in these individuals’ genomes. Instead we aimed to demonstrate to what extent, in principle, any individual can interpret their personal genome using only public resources with an affordable budget and no laboratory equipment. We stress that our goal is not to diminish the value of DTC industry services, which have catalyzed the access to, and interest in, personal genomics data in the wider public. However, as the adoption of DTC personal genomics tests becomes ever more widespread, we envisage community phenotype association and third party public tools to become more significant in the overall interpretation of personal genomics results.\n\nAlthough we found the no-call rate to be comparable between both versions of the 23andMe platform, Mother had a greater undercall rate than the other individuals. Also, results given by no-call rates suggest that there might be some intrinsic differences between the two chips. Nevertheless the current number of samples analyzed is not large enough to make this conclusion, as only five chips of data analyzed do not provide any basis for their overall performance. Therefore our error estimation rates presented here should only be considered in the context of this analysis and not as representative of the DTC company’s overall quality scores.\n\nBased on the no-call rate and the relative ratios of homozygous and heterozygous sites reported, we computed an ‘undercall’ rate and used it to estimate the number of heterozygous sites mistakenly reported as homozygous. The frequency of such events is small (~0.2%). Nevertheless, the fact that up to 2,000 heterozygous variants may be missed is a reminder that interpretation of personal disease risks should be done with caution. Findings of potential medical relevance should always be verified for correctness. Wherever possible, it is beneficial to perform the analysis in the context of families: identification of MIEs and State Consistency Errors10 is a powerful tool to assess genotyping quality.\n\nThe similar level at which identical genotypes is shared between the relatives and the non-CEU individual is consistent with the ethnically close background for the family members. This, however, does not suggest that Mother or Father are directly related. In fact, when performing an admixture analysis with unrelated individuals of Southern European descent (Figure 2) it is clear that while the parents cluster within reasonably close distance to other Spanish individuals, they display a typical level of genotype sharing between two people from the Iberian Peninsula.\n\nWe also indicated that when comparing genotype similarities between siblings and parents, we found that Daughter was significantly closer to Father than Son was. Although the expectation was that both Daughter and Son should be equally similar to both parents, these unexpected results may be the reflection of bias in the subset of markers used in the DTC analysis. These results are not therefore indicative of Daughter’s genome being closer to either parent, as most of the genome is missing and hence any inference in this respect cannot be made. In the context of SNP analysis, however, it is worthwhile reporting such SNP differences as these will influence the overall results reported back to the DTC customer. This in turn may explain why observed susceptibility risks vary among family members when comparing their phenotypic annotations.\n\nThe identification of blocks of identical, haploidentical or nonidentical genotype between family members (e.g. Son and Daughter in Figure 4), highlights the location of meiotic recombinations. These blocks provide well defined expectations for whether the genes included in them should display similar or distinct genotypes. This information is valuable in the context of genetic research10 but also to the general public, to predict shared phenotypes among family members. Within ‘identical’ blocks, siblings are essentially identical twins: this fact gains special personal meaning in the context of DTC genetic analysis.\n\nPublicly curated data, like that available in SNPedia, is exposed to error due to human mistakes or malicious intent. Although this is a legitimate concern, it has been shown that with similar public annotation resources (Wikipedia for example), vandalism rates are very low in areas of specialist academic interest20. As these open access resources mature and grow, maintaining accuracy will be an important consideration for the contributing community.\n\nIn this study we have not found any annotated genotype that is likely to raise significant health concerns among the family individuals. It is inevitable, however, that as more individuals investigate their own personal genomes, and more statistical associations are uncovered, genotype/phenotype correlations with serious health implications will be accessible through open access resources. Interpreting such information appropriately is bound to be difficult for individuals who are not expert geneticists; this poses special ethical challenges from the point of view of how to present the data. DTC companies have recognized this ethical issue by implementing additional access controls to some particularly sensitive annotations, such as those genotypes associated with a high risk of developing Alzheimer’s disease or some forms of breast cancer. To our knowledge, public annotation resources do not currently make such distinctions. We therefore urge caution when investigating personal genomic data for health risks, especially when using open access information. We recommend those who uncover personal genomic information of medical concern seek the advice of genetic counselors, who can interpret and advise on the context of what it really means to the tested individual.\n\n\nMethods\n\nFive 23andMe genome analysis kits were purchased at two time points. Son’s kit was bought in May 2009 and was tested with 23andMe version 2 (~576,000 SNPs). The other 4 family members were analyzed in one batch, with kits bought in December 2010 and results returned in February 2011. The second batch used 23andMe’s chip version 3 with ~967,000 SNPs per genome analyzed. After discussion of results, consent was given by all family members of the family to publish their genotypes.\n\nAssume N SNPs were tested, and of these, fN are truly heterozygous. We wished to compute f (heterozygous fraction) from the observed numbers of homozygous, heterozygous and failed SNPs.\n\nFor each SNP with a dbSNP ‘rs’ identifier, we assumed that 1) the SNP is biallelic, 2) it is present in diploid state. We further assumed a probability x of not observing a given allele (the \"undercall rate\"), and assumed this probability was equal for both alleles, at all sites. Finally, we assumed that the probability of observing a wrong allele was zero (\"overcall rate\").\n\nIf the true state of the SNP was heterozygous, the following could have happened. 1) If neither allele was observed (double undercall), the SNP was called \"NULL\" (with conditional probability = x2). 2) If one allele was not observed (single undercall), the SNP was called \"HOM\" (conditional probability = 2x). 3) If both alleles were observed, the SNP was called \"HET\" (conditional probability = 1-2x-x2).\n\nIf the true state of the SNP was homozygous, there was only one type of allele to be observed. Thus, the following could have happened. 1) If the allele was not observed, the SNP was called \"NULL\" (conditional probability = x). Otherwise, 2) the SNP was called \"HOM\" (conditional probability = 1-x).\n\nThe expected frequencies for NULL, HET and HOM were:\n\nNULL’ = fx2+(1-f)x\n\nHET’ = f(1-2x-x2)\n\nHOM’ = (1-f)(1-x)+2fx = 1-x+f(3x-1)\n\nSince HOM + HET + NULL = 1, the undercall rate x was given by solving:\n\nx3 + (1+HOM)x2 + (3HET+2HOM-3)x + NULL = 0\n\nThe heterozygous fraction f was then given by: f = HET/(1-2x-x2), and the number of \"missing\" heterozygous sites could be computed by: missing = (f-HET)N. Finally, the number of heterozygous sites reported as homozygous was given by:\n\nhet2hom = missing - N*NULL*HET/(HET+HOM)\n\nRaw reads were aligned to the reference GRCh37 using bwa 0.6121. Local realignment was performed around indels with the Genome Analysis Toolkit (GATK v1.4)15 framework for variation discovery and genotyping using next-generation DNA sequencing data. Optical and PCR duplicates were marked in BAM files using Picard 1.6214. Original HiSeq base quality scores were recalibrated using GATK TableRecalibration and variants called with GATK UnifiedGenotyper. Indels and SNPs were hard-filtered according to Broad Institute best-practice guidelines22 to eliminate false positive calls and produce the final VCF.\n\n\n\nPromethease, a SNPedia tool for phenotype inference, was used for assignment of SNPedia annotations to observed SNPs. SNPedia annotations contain manually curated SNPs that summarize phenotype associations observed in a particular population. Phenotype associations were inferred using SNPedia’s SNP ids, which correspond to dbSNP23. In our analysis only SNPedia annotations of >= 3 Magnitude were examined. Magnitude is a subjective score that helps prioritize SNPs according to their expected importance and the phenotypic annotation itself in the form of free text. Magnitude is assigned by SNPedia entry curators.\n\nWe calculated similarity scores using our own Perl scripts and MySQL. Similarity between any given two individuals was calculated as the total number of matching SNPs plus half-matches divided by 2. Negative results were counted as the total number of conflicts plus the number of half-matches divided by 2. As the platforms used are different for Son as compared to the rest, Son can only be compared relative to himself and not against other individuals. Similarities between individuals were statistically tested using the R package for Pearson’s Chi-squared with Yates’ correction.\n\n\nConsent\n\nWritten consent for publication of their genotype and phenotype was obtained from all family individuals.",
"appendix": "Author contributions\n\n\n\nManuel Corpas and Darren Logan conceived the experiments. Manuel Corpas, Gustavo Glusman and Darren Logan wrote the paper with input from the other authors. Gustavo Glusman, Mike Cariaso, Manuel Corpas, Daniel Swan contributed with data and analyses, Rafael Jimenez, Bastian Greshake and Jong Bhak contributed comments/suggestions and their expertise of the field. All authors read and approved the final version.\n\n\nCompeting interests\n\n\n\nMichael Cariaso is the developer of Promethease and SNPedia. Some aspects of the Promethease analysis shown here cost $2 per individual.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful to the Eurogenes Genetic Ancestry Project for providing the admixture analysis of individuals from (Southern Europe Figure 2).\n\n\nReferences\n\nSaxena R, Voight BF, Lyssenko V, et al.: Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science. 2007; 316(5829): 1331–1336.PubMed Abstract | Publisher Full Text\n\nPhillips C, Salas A, Sánchez JJ, et al.: Inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs. Forensic Sci Int Genet. 2007; 1(3–4): 273–280.PubMed Abstract | Publisher Full Text\n\nCariaso M, Lennon G: SNPedia: a wiki supporting personal genome annotation, interpretation and analysis. Nucleic Acids Res. 2012; 40(Database issue): D1308–D1312.PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttp://opensnp.org/\n\nhttp://www.mendeley.com/\n\nHuss JW III, Lindenbaum P, Michael M, et al.: The Gene Wiki: community intelligence applied to human gene annotation. Nucleic Acids Res. 2010; 38(Database issue): D633–D639.PubMed Abstract | Publisher Full Text | Free Full Text\n\nGood BM, Clarke EL, Loguercio S, et al.: Linking genes to diseases with a SNPedia-Gene Wiki mashup. J Biomed Semantics. 2012; 3(Suppl 1): S6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGibbs RA, Belmont JW, et al.: The international HapMap project. Nature. 2003; 426(6968): 789–796.PubMed Abstract | Publisher Full Text\n\nhttp://www.23andme.com/\n\nRoach JC, Glusman G, Smit AF, et al.: Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science. 2010; 328(5978): 636–639.PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttp://customercare.23andme.com/entries/21259007-what-does-no-call-not-genotyped-mean-in-browse-raw-data\n\nAlexander DH, Novembre J, Kenneth L, et al.: Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 2009; 19(9): 1655–1664.PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttp://bga101.blogspot.com/\n\nhttp://picard.sourceforge.net/\n\nMcKenna A, Hanna M, Eric B, et al.: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9): 1297–1303.PubMed Abstract | Publisher Full Text | Free Full Text\n\nNg PC, Henikoff S: SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 2003; 31(13): 3812–3814.PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdzhubei IA, Schmidt S, Leonid P, et al.: A method and server for predicting damaging missense mutations. Nat Methods. 2010; 7(4): 248–249.PubMed Abstract | Publisher Full Text | Free Full Text\n\nGonzalez-Perez A, Lopez-Bigas N: Improving the assessment of the outcome of nonsynonymous SNVs with a consensus deleteriousness score, Condel. The American Journal of Human Genetics. 2011; 88(4): 440–449.Publisher Full Text\n\nhttp://www.snpedia.com/index.php/Promethease\n\nFinn RD, Gardner PP, Bateman A, et al.: Making your database available through Wikipedia: the pros and cons. Nucleic Acids Res. 2012; 40(Database issue): D9–D12.PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–1760.PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttp://www.broadinstitute.org/gsa/wiki/index.php/Best_Practice_Variant_Detection_with_the_GATK_v3\n\nSherry S, Ward MH, Kholodov M, et al.: dbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 2001; 29(1): 308–311.PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "200",
"date": "23 Jul 2012",
"name": "Peter N. Robinson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n23andMe and several other companies have been offering Direct-to-consumer (DTC) genomic testing for several years now. In general, such tests seem to have a bad reputation amongst human geneticists because of the perception that individuals may not be able to interpret the findings in a useful way.Additionally, although genetic testing has an important and widely accepted role in the diagnostics of Mendelian disease and several other areas, many feel that the clinical utility of genomic testing for common disorders such as heart disease or hypertension has not been shown to date. For instance, the Evaluation of Genomic Applications in Practice and Prevention Working Group (EWG) found insufficient evidence to recommend testing for the 9p21 genetic variant or 57 other variants in 28 genes to assess risk for cardiovascular disease in the general population (Genetics in Medicine (2010) 12, 839–843).Be that as it may, has become well known and the range of variants now tested by companies such as 23andMe is increasing; with the advent of relatively cheap exome and even genome sequencing, it seems quite likely that DTC exome and genome sequencing will be offered in the not too distant future. Therefore, it is important to understand several aspects about DTC testing, including the quality of the raw product (i.e., are the genotype calls correct), the depth, correctness, and utility of annotations provided by DTC companies or widely available to the public, and perhaps most importantly, to know how typical consumers of DTC products use the findings for their own health care or, say, for planning life-style modifications.This paper offers a detailed look at several of these aspects, and I think does an excellent job at providing the reader with a sense of what kind kind of data consumers of DTC products can expect. I think the paper would profit from a number of minor revisions. The paper essentially deals with two different topics. The first involves a number of computational quality control procedures that require a good deal of bioinformatics expertise (I doubt that much of this would be in the reach of most of the customers of DTC sequencing), ranging from a calculation of error rates, an analysis of Mendelian inheritance errors, genotype distributions, and admixture analysis. It was not entirely clear to me how the p-values reported for the SNP similarity analysis were calculated, and a more precise definition of how pseudo-MIEs were counted should be provided. The authors should provide full methodological details of how this analysis was performed. It is also something of a distraction that one of the samples was analyzed using the 23andMe v2 kit, while the rest of the family was analyzed using the v3 kit. I imagine simply that the Son was the person who “went first”, but the apparently different error characteristics of the two versions limit somewhat the findings of the paper.The second major aspect of this paper involves analysis that could be done by non-specialists, using tools such as SNPedia. It was not entirely clear to me why the authors performed the analysis in Figure 5. The heading called “Inference of Phenotypes Using SNP data” is misleading because actually what the findings reveal is a genotype, say, a risk for going bald. However, if the son is not currently bald, then he does not now have the phenotype “baldness”. Instead, the only thing that can be inferred from the genotype is an increased risk of baldness. This is a very important distinction, and it is very important especially for the general public to realize that having a genotype that is associated with an increased risk of some phenotype does not necessarily mean that one will actually develop that phenotype.It would be a nice addition to this paper to hear more about how the family reacted to these findings. Have the increased risks for some of the diseases mentioned led to life style changes? Did the family members report the findings to their physicians? Was a family member worried or uncomfortable about hearing the findings?",
"responses": []
},
{
"id": "201",
"date": "26 Jul 2012",
"name": "Christian Gilissen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written and interesting article that describes analyses on genome-wide genotyping information from a DTC company.Major concernsAlthough the analyses are carried out very well, the overall purpose and conclusion of the paper is rather mixed and somewhat unclear. The authors state that they “explore the extent to which phenotype inference and genotype analysis can be carried out solely using existing public or very low cost resources.” or as in the abstract “providing an evaluation of the current potential of public available analysis tools for personal genomics”.The discussion section focuses very much on the results of the different analyses, whereas that is (according to the purpose of the paper) not that relevant. Rather in that context it would be more interesting to speculate on other aspects (for example, how easy it is for a typical DTC customer to apply the publicly available tools, or how easy it is to (correctly) interpret the outcome of such tools) and to provide a real conclusion on “the extent to which phenotype inference and genotype analysis can be carried out solely using existing public or very low cost resources.”It is not clear whether the tools that were investigated compromise all public domain tools for these kinds of analyses. If the authors do not investigate all available tools, the authors should motivate their choice for the described toolsGiven the purpose of the paper, a table with the available tools and their “potential” would be very useful.Minor concernsThe fact that “Son” was done with a V2 assay complicates all comparisons in the paper. It seems to paper would be a lot more straightforward if the authors used a V3 assay for the son as well.Page 7, “Exome data summary statistics”, authors mention that the numbers they find are what is to be expected. Please add a reference to substantiate this.Page 10, the cond. prob. of a heterozygous call for a heterozygous SNP reads “1-2x-x2” and should probably be “1-2x-x^2”. Similar with the expected frequency for NULL’.SummaryIs the title appropriate for the content of the article? YesIs the abstract a suitable summary of the article? YesIs the article well constructed and clear? Yes, the article is well constructed, but could do with a conclusion and discussion that is more in line with the purpose of the paper. (see comments above)Is there adequate analysis, including information on how the data were analyzed (e.g. programs, code, stats etc.)? YesAre the conclusions sensible and balanced? The conclusions does not answer the questions that were asked at the outset of the paper.Have any potential biases or competing interests been disclosed? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-3
|
https://f1000research.com/articles/1-1/v1
|
13 Jul 12
|
{
"type": "Research Article",
"title": "Knowledge of specific HIV transmission modes in relation to HIV infection in Mozambique",
"authors": [
"Devon D Brewer"
],
"abstract": "Background: In prior research, Africans who knew about blood-borne risks were modestly less likely to be HIV-infected than those who were not aware of such risks.Objectives/Methods: I examined the association between knowledge of specific HIV transmission modes and prevalent HIV infection with data from the 2009 Mozambique AIDS Indicator Survey.Results: Respondents displayed high awareness of blood exposures and vaginal sex as modes of HIV transmission. However, only about half of respondents were aware of anal sex as a way HIV can be transmitted. After adjustments for demographics and sexual behaviors, respondents who knew that HIV could spread by contact with infected blood or by sharing injection needles or razor blades were less likely to be infected than those who did not know about these risks. Respondents who knew about sexual risks were as, or more, likely to be HIV infected as those who did not know about sexual risks. Also, children of HIV-uninfected mothers were less likely to be infected if their mothers were aware of blood-borne HIV risks than if their mothers were unaware.Conclusion: HIV education campaigns in Mozambique and elsewhere in sub-Saharan Africa should include a focus on risks from blood exposures and anal sex.",
"keywords": [
"iatrogenic disease",
"HIV",
"knowledge",
"health education",
"infectious disease transmission"
],
"content": "Introduction\n\nA wide variety of blood exposures is independently associated with incident and prevalent HIV infection in sub-Saharan Africa1–9. Anal sex has also been repeatedly associated with HIV infection in heterosexual Africans10–15, with some exceptions16,17. Knowledge of blood-borne HIV risks varies markedly across the region, with awareness much higher in West, central and East Africa than in southern Africa18. This geographic variation in knowledge corresponds closely to differences in HIV prevalence, such that countries in which many people know about blood-borne risks have much lower HIV prevalence than countries in which few know about these risks. Although HIV education campaigns in West, central and East Africa have included a focus on blood-borne risks, campaigns in Southern Africa have not, likely leading to deficits in Southern Africans’ knowledge. At the individual level, persons who know about blood-borne risks are modestly less likely to be HIV-infected than those who are not aware of such risks, independent of demographics and sexual behaviors18.\n\nIn contrast, belief in condom use as a strategy for avoiding HIV infection is positively associated with HIV infection at both the individual and national levels in sub-Saharan Africa18. That is, those who believe condoms are an effective strategy for preventing HIV transmission are more likely to be HIV-infected than those who do not believe condom use protects against HIV, independent of demographics and sexual behaviors.\n\nAlthough long assumed to be rare, anal sex is not uncommon in sub-Saharan Africa, as modest to large proportions of heterosexual men and women report engaging in it10–15,19–27. Research based on nonprobability samples suggests that many Africans are unaware that anal sex is a significant risk for HIV transmission. While 87% of young adult Nigerians in one study knew anal sex is an HIV risk28, only 54% of men in Cape Town, South Africa in 199919 and 29% of adolescents in a Western Cape (South Africa) township with very high HIV prevalence shared that perception26. Some South African women in a qualitative study explained the lack of risk involved with anal sex29. Moreover, Kenyan prostitutes viewed vaginal sex as equally or more risky than anal sex21. Throughout the last 13 years, researchers have noted that HIV education campaigns targeting heterosexuals in sub-Saharan Africa lack warnings about the risk of anal sex10,15,19,23,24,27,30.\n\nIn this paper, I report on the relationship between knowledge of HIV transmission routes and prevalent HIV infection in Mozambique, a country with high HIV prevalence not included in prior analyses. I analyzed data from the 2009 Mozamibique AIDS Indicator Survey (AIS), a special version of the Demographic and Health Surveys (DHS) conducted in poor countries. The 2009 Mozambique AIS differs from prior DHS with respect to how knowledge of HIV transmission risks was measured. In the 2009 Mozambique AIS respondents were asked directly about several specific blood-borne and sexual transmission routes. The 2009 Mozambique AIS also involved HIV testing of children, enabling examination of the association between mothers’ knowledge of blood-borne HIV transmission routes and HIV infection in their children.\n\n\nMethods\n\nIn the 2009 Mozambique AIS, the household participation rate was 99%, and 95% of eligible women (age 15-64) and 90% of eligible men (age 15–64) participated31. Ninety-two percent of adult respondents and 87% of children between ages 0 and 11 years provided dried blood spot specimens for HIV testing. HIV serostatus was determined with an enzyme-linked immunosorbent assay (ELISA) test and confirmed by a different ELISA test. Specimens with discrepant ELISA results were confirmed by a further ELISA test. The 2009 Mozambique AIS data and documentation are available at http://www.measuredhs.com.\n\nI included respondents’ data in analysis if they reported never having been tested previously for HIV and also reported awareness of HIV/AIDS. HIV counseling may, in some places, involve education about blood-borne and sexual risks32, which may confound the relationship between knowledge and serostatus33. Respondents who did not report awareness of HIV/AIDS were not asked about ways that it could be transmitted. Ninety-seven percent of female respondents and 99% of male respondents reported they had heard of HIV/AIDS.\n\nRespondents who reported awareness of HIV/AIDS were asked whether it was possible to transmit HIV through each of several types of direct and indirect interpersonal contact. I focused on knowledge of four types of contact: sharing injection needles/blades; contact with infected blood; unprotected vaginal sex (i.e., vaginal sex without a condom); and unprotected anal sex. I coded \"don’t know\" responses to these questions as \"no\".\n\nFor the analyses of mother’s knowledge and child’s serostatus, I included biological mother-child pairs in which the mother was HIV-uninfected, to eliminate confounding by vertical transmission.\n\nIn the 2003 Mozambique DHS, the household participation rate was 95%, and 91% of eligible women (age 15-49 years) and 81% of eligible men (age 15–64 years) participated34. Women were recruited as respondents from all households, but men were recruited as respondents only from one-third of households. Respondents did not provide blood specimens for HIV testing. The 2003 Mozambique DHS data and documentation are also available at http://www.measuredhs.com.\n\nAs I did with the AIS data, I included respondents’ data in analysis if they reported never having been tested previously for HIV and also reported awareness of HIV/AIDS. Ninety-seven percent of female respondents and 99% of male respondents in the 2003 DHS reported they had heard of HIV/AIDS.\n\nRespondents who reported awareness of HIV/AIDS were asked \"Do you know how you can avoid HIV/AIDS?\" Those answering affirmatively were then asked \"How can you prevent HIV/AIDS?\" Interviewers encouraged and recorded multiple responses, and responses were coded by DHS staff. To compare with the 2009 Mozambique AIS and other DHS, I focused on two measures: whether a respondent had a response that was coded as \"only use sterilized syringes/needles\" and \"always use a condom\". I coded \"don’t know\" responses to the open-ended question as \"no\" for each of these measures.\n\nWith the 2009 Mozambique AIS data, I computed cross-classifications, odds ratios, and the associated 95% confidence intervals (CI) for the relationships between each knowledge measure and prevalent HIV infection, separately for women and men. To examine the associations further, I calculated the odds ratios, with HIV status as the dependent variable and a knowledge measure as the independent variable, adjusted for age (in whole years), urban/rural residence, number of whole years of education, wealth (in quintiles), reported number of sex partners in the prior 12 months, and whether the respondent reported having had a sexually transmitted disease in the prior 12 months. In addition, I calculated adjusted odds ratios (AORs) in the manner just described but adjusting for each of the three other knowledge measures as well. I also computed the Pearson (phi) correlation between each pair of knowledge measures. I used SPSS 7.5 (SPSS Inc., Chicago, USA) to perform the analyses. I compared the adjusted odds ratios for Mozambique with similar DHS results from four countries in Southern Africa (Lesotho, Malawi, Swaziland, and Zimbabwe) based on knowledge measures derived from free responses to an open-ended question about ways to avoid HIV infection18.\n\nIn July of 2011, I sought evidence of public HIV education campaigns focused on blood-borne risks in Mozambique by searching two online databases: the Google search engine and the Media/Materials Clearinghouse. For the Google search, I used the key words \"Mozambique\", \"HIV\", and \"razor\". I examined the resources identified until I found 30 consecutive resources to be irrelevant (for a total 320 resources examined). I repeated this search in September of 2011 at the Google’s Mozambican site, with the equivalent key words in Portuguese: \"Moçambique\", \"VIH,\" and \"lâmina\" (290 resources examined). I also extended this search by adding, in turn, the names of the 14 HIV prevention programs and campaigns mentioned in the AIS questionnaire (for questions about respondents’ familiarity with the programs and campaigns)31. My Google search was not exhaustive, especially given the limited scope induced by the key word \"razor\". However, the razor blade is perhaps the most ubiquitous sharp instrument involving possible blood exposure in daily life in Mozambique. Any public education campaign about blood-borne HIV risks that excludes explicit mention of razor blades is probably superficial or very narrow. For the Media/Materials Clearinghouse search, I inspected all materials pertaining to Mozambique. I also examined any sources related to Mozambique that I had found in an earlier search of the US National Library of Medicine Gateway18.\n\n\nResults\n\nIn the 2009 Mozambique AIS, over 90% of respondents reported that HIV could spread through shared needles/razors and nearly 80% reported that HIV could be spread through contact with infected blood (Table 1). Approximately 90% of women and men reported vaginal sex as an HIV transmission route. In contrast, only 48% of women and 58% of men thought that HIV could spread through anal sex.\n\nBefore adjustments for demographics and sexual behavior variables, respondents who believed that HIV spread through vaginal or anal sex were more likely to be infected than those who did not believe HIV spread in that way (Table 1). In unadjusted analyses, respondents who reported that HIV could be transmitted through blood contact or reusing sharps were about as likely to be infected as respondents who did not think HIV was transmitted in that way.\n\nUrban residence, education, and wealth were moderately positively associated with knowledge of blood-borne HIV risks (results not shown), and these same factors were positively related to HIV infection as well31. Consequently, the AORs between the knowledge measures and HIV infection in (Table 2 and Table 3) provide better estimates of the relationship between the knowledge measures and HIV infection.\n\nTable 2 Note: 95% confidence intervals in parentheses\n\n1Adjusted for age, urban/rural residence, education, wealth, reported number of sex partners in the prior 12 months, and reported sexually transmitted disease in the prior 12 months\n\n2Summary of results from Lesotho, Malawi, Swaziland, and Zimbabwe for similar knowledge measure\n\n3Includes same adjustments as for single measure AOR, but also adjusted for the 3 other knowledge measures\n\nTable 3 Note: 95% confidence intervals in parentheses\n\n1Adjusted for age, urban/rural residence, education, wealth, reported number of sex partners in the prior 12 months, and reported sexually transmitted disease in the prior 12 months\n\n2Summary of results from Lesotho, Malawi, Swaziland, and Zimbabwe for similar knowledge measure\n\n3Includes same adjustments as for single measure AOR, but also adjusted for the 3 other knowledge measures\n\nAfter adjustments for demographic factors and sexual behavior variables, the measures of knowledge of blood-borne risks become inversely related to HIV infection. That is, respondents who reported that HIV can spread through shared needles/razors and blood contact were less likely to be infected than those who reported that HIV does not spread through such means, once demographics and sexual behaviors were held constant. The adjustments for potential confounders also caused the positive associations for knowledge of vaginal sex risk in women and knowledge of anal sex risk in men to disappear. However, the adjusted associations for knowledge of anal sex risk in women and knowledge of vaginal sex risk in men remained meaningfully positive. In particular, men who believed HIV spreads through vaginal sex were almost twice as likely to be infected as men who did not believe this (Table 3).\n\nThe adjusted odds ratios for knowledge of shared needles/razors as transmission risks are generally similar to those for knowledge of avoiding shared razors as an HIV prevention strategy in four other countries in Southern Africa (Table 2 and Table 3). The adjusted associations for knowledge of vaginal sex risk also approximate those for knowledge of condoms as a prevention strategy from the other Southern African countries.\n\nThe blood-borne risk knowledge measures correlated moderately with each other (r = 0.26–0.29) as did the sexual risk knowledge measures (r = 0.23–0.29). The associations between one blood-borne risk knowledge measure and one sexual risk knowledge measure were weaker (r = 0.12–0.23). Nonetheless, when all four knowledge measures were included in analysis together (last column in (Table 2 and Table 3), the associations between each knowledge measure and HIV infection were almost the same as when the knowledge measures were analyzed separately.\n\nHIV uninfected mothers’ knowledge of blood-borne HIV risks was also inversely related to their children’s HIV status. Children whose mothers knew that contact with blood is an HIV risk were less likely to be infected (0.4%; 11/2511) than children whose mothers did not know about this risk (1.2%; 7/591; OR 0.37, 95% CI 0.14–0.95). Similarly, children whose mothers knew that sharing needles and razors was a risk were also less likely to be infected (0.5%, 16/2918) than children whose mothers were unaware of this risk (1.1%, 2/185; OR 0.50, 95% CI 0.12–2.21).\n\nIn the 2003 Mozambique DHS, 9.8% (1110/11381) of women and 18.9% (413/2709) of men recalled \"only use sterilized syringes/needles\" as a way to prevent HIV. However, 55% (6253/11381) of women and 68% (1845/2709) of men recalled \"always use a condom\" as a way to prevent HIV.\n\nThere was no online evidence of public HIV education campaigns that focused on blood-borne transmission risks in Mozambique. Some participants in a face-to-face community HIV education program that was implemented in Mozambique credited the program with informing them about some blood-borne risks35,36. However, none of the descriptions of the program at the program’s website (http://www.steppingstonesfeedback.org) or elsewhere online indicate that this topic is formally covered in the program.\n\n\nDiscussion\n\nAdult Mozambicans displayed very high awareness of some blood exposures and vaginal sex as modes of HIV transmission when directly asked about such contact in the 2009 Mozambique AIDS Indicator Survey. However, only a little more than half of respondents were aware of anal sex as a way HIV can be transmitted. After adjustments for demographics and sexual behaviors, knowledge of blood-borne risks was inversely associated with prevalent HIV infection. That is, respondents who knew that HIV could spread by contact with infected blood or by sharing injection needles or razor blades were less likely to be infected than those who did not know about these risks. Knowledge of sexual risks was negligibly to positively related to prevalent HIV infection. The positive association was strongest in men: those acknowledging unprotected vaginal sex as a risk were almost twice as likely to be infected with HIV as those not acknowledging. These associations between knowledge of blood-borne and sexual risks and prevalent HIV infection in Mozambique are similar in magnitude to those observed in four other Southern African countries based on roughly parallel knowledge measures. In addition, mothers’ knowledge of blood-borne risks was inversely associated with HIV infection in their children, which suggests that persons aware of such risks not only are more likely to take corresponding preventive measures for themselves but for those in their care as well. Moreover, there was no online evidence that public HIV education campaigns in Mozambique have included a meaningful focus on blood-borne risks.\n\nThe very high proportion of Mozambicans who were aware of blood-borne HIV risks (approximately 90%) in the 2009 AIS contrasts sharply with the very low proportions of 2003 Mozambique DHS respondents and DHS respondents in four other Southern African countries who mentioned such risks (8–23%)18. This difference seems to be due to assessing knowledge in the 2009 Mozambique AIS by recognition of specific risks and in the other DHS by recall of risks. In South Africa and Swaziland, the discrepancies between levels of knowledge of blood-borne HIV risks when measured by recognition18,26,37 and recall18,38–42 are even larger. These combined results refute my earlier hypothesis18 that Southern Africans and perhaps Africans generally know that contaminated blood can transmit HIV only when the exposure is visible and large. Instead, these results indicate that Southern Africans are typically aware of a variety of blood-borne HIV risks (including shared needles and razors that might not involve large or visible blood contamination), but only when specifically prompted.\n\nInterestingly, observed levels of Southern Africans’ knowledge of sexual risks differ relatively little based on measurement approach. In the aforementioned studies, the proportions of respondents aware of sexual HIV transmission risks tended to be only slightly less when measured with recall methods than recognition methods. The difference between the proportion of 2003 Mozambique DHS respondents who recalled \"always use a condom\" as a prevention strategy (62%) and the proportion of 2009 Mozambique AIS respondents who recognized vaginal sex as a transmission mode (92%) is somewhat larger, although this could be due to increases over time in Mozambicans’ perceptions of vaginal sex as a risk.\n\nThe discrepancy between observed levels of knowledge of blood-borne HIV risks based on recall and recognition measurement reveals that blood-borne risks are not salient in the minds of Southern Africans. In 2011, Class interviewed 23 HIV infected adults and 26 parents or other caretakers of HIV infected children in Maputo, Mozambique (Deena Class, personal communications, July 11, 2011, August 27, 2011, and September 29, 2011). Nearly all of Class’ respondents noted blood-borne transmission as one mode of HIV spread. However, none of the adults attributed their infections to a blood exposure. Five of the infected children had seronegative biological mothers, indicating that these children had acquired their infections horizontally. Remarkably, only one of these mothers suspected blood-borne transmission for her child. Furthermore, none of these five mothers were told by their healthcare providers how their children might have been infected. In another study, Zimbabwean youth almost universally recognized the potential for HIV transmission through shared razors, yet ranked the avoidance of sharing razors as the least effective strategy for preventing HIV in comparison to strategies focused on sexual exposures and behaviors not known to protect against infection (avoiding deep kissing and washing hands)43. Similarly, Kenyans in Nyanza province grossly underestimated how long HIV can survive on a blood-contaminated razor blade or inside a blood-contaminated needle or syringe44. Thus, many Africans may recognize blood-borne HIV transmission as a theoretical possibility, but regard it as unlikely in practice.\n\nSuch apparent misunderstandings of blood-borne HIV transmission are not surprising given the lack of public education about blood-borne risks in sub-Saharan Africa. Just as in other African countries18, efforts to educate Mozambican traditional healers about hygienic practices began decades ago45,46 and continue to this day47,48. There was also at least one attempt to educate barbers about blood-borne risks in the early 1990s49. However, I found no good evidence that public HIV education campaigns in Mozambique have in the past focused on blood-borne risks to any meaningful extent. Furthermore, as in other Southern African countries18, large recent and current public HIV education campaigns in Mozambique exclude mention of blood-borne risks50,51 (see also: One Love Regional Campaign – Southern Africa, and Center for Communication Programs). Indeed, Class’ Mozambican respondents who noted blood-borne transmission as one mode of HIV spread said they learned of these risks informally, not from HIV education campaigns, health care providers, or public health sources (Deena Class, personal communication, September 29, 2011).\n\nMozambicans’ awareness of anal sex as a transmission risk was much lower than their awareness of vaginal sex as a risk, even though the actual risk from anal sex is much higher. Contrary to my expectation, knowledge of anal sex risk was not associated with lower rate of HIV infection. It is unclear why this is so. Further investigation of the perceived risk of anal sex (e.g., whether risk differs for men and women) and whether respondents engage in anal sex might help clarify this result.\n\nBeliefs in vaginal sex as an HIV transmission risk and condom use as an HIV prevention strategy18 are associated with a higher likelihood of HIV infection in Mozambique and elsewhere in sub-Saharan Africa. This is consistent with the lack of association between condom use in vaginal sex and incident HIV infection14,52,53. Condom use itself may even make people more vulnerable to infection by reducing mucosal exposure to HIV and thus hindering the development of alloimmunity against HIV54,55.\n\nTo determine the modes of transmission of HIV in sub-Saharan Africa with confidence, researchers must assess blood and sexual exposures comprehensively in incident HIV cases and controls, trace their contacts corresponding to such exposures, and sequence the DNA in infected persons’ HIV isolates56–58. In the meantime, HIV education campaigns in Mozambique and other poor countries should focus on blood-borne and anal sex transmission risks comprehensively and emphasize strategies for avoiding these dangers59–61.",
"appendix": "Competing interests\n\n\n\nNo relevant competing interests declared.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nI thank Stuart Brody, Deena Class, David Gisselquist, and John Potterat for helpful comments on an earlier draft of this article. I also thank the government of the Republic of Mozambique for making the data from the 2009 Mozambique AIS and 2003 Mozambique DHS publicly available.\n\n\nReferences\n\nSt Lawrence JS, Klaskala W, Kankasa C, et al.: Factors associated with HIV prevalence in a pre-partum cohort of Zambian women. Int J STD AIDS. 2006; 17(9): 607–613. PubMed Abstract | Publisher Full Text\n\nDeuchert E: Maternal health care and the spread of AIDS in Burkina Faso and Cameroon. World Health Pop. 2007; 9(3): 55–72. PubMed Abstract | Publisher Full Text\n\nDeuchert E, Brody S: The role of health care in the spread of HIV/AIDS in sub-Saharan Africa: evidence from Kenya. Int J STD AIDS. 2006; 17(11): 749–752. PubMed Abstract | Publisher Full Text\n\nBrewer DD, Potterat JJ, Roberts JM Jr, et al.: Male and female circumcision associated with prevalent HIV infection in virgins and adolescents in Kenya, Lesotho, and Tanzania. Ann Epidemiol. 2007; 17(3): 217–226. PubMed Abstract | Publisher Full Text\n\nGisselquist D: Points to consider: Responses to HIV/AIDS in Africa, Asia, and the Caribbean. Adonis and Abbey Publishers Ltd, London; 2007. Reference Source\n\nPeters EJ, Brewer DD, Udonwa NE, et al.: Diverse blood exposures associated with incident HIV infection in Calabar, Nigeria. Int J STD AIDS. 2009; 20(12): 846–851. PubMed Abstract | Publisher Full Text\n\nOkinyi M, Brewer DD, Potterat JJ, et al.: Horizontally acquired HIV infection in Kenyan and Swazi children. Int J STD AIDS. 2009; 20(12): 852–857. PubMed Abstract | Publisher Full Text\n\nVaz P, Pedro A, Le Bozec S, et al.: Nonvertical, nonsexual transmission of human immunodeficiency virus in children. Pediatr Infect Dis J. 2010; 29(3): 271–274. PubMed Abstract | Publisher Full Text\n\nBrewer DD: Scarification and male circumcision associated with HIV infection in Mozambican children and youth. WebmedCentral EPIDEMIOLOGY 2011, 2(9): WMC002206. Reference Source\n\nLane T, Pettifor A, Pascoe S, et al.: Heterosexual anal intercourse increases risk of HIV infection among young South African men. AIDS. 2006; 20(1): 123–125. PubMed Abstract | Publisher Full Text\n\nMlotshwa M, Abdool Karim Q, Frohlich J, et al.: Risk factors for HIV infection in young women in rural South Africa: implications for HIV prevention trials. Abstract MOPE0276, 17th International AIDS Conference, Mexico City, August, 2008. Reference Source\n\nGrijsen ML, Graham SM, Mwangome M, et al.: Screening for genital and anorectal sexually transmitted infections in HIV prevention trials in Africa. Sex Transm Infect. 2008; 84(5): 364–370. PubMed Abstract | Publisher Full Text\n\nAdoga MP, Banwat EB, Forbi JC, et al.: Human immunodeficiency virus, hepatitis B virus and hepatitis C virus: sero-prevalence, co-infection and risk factors among prison inmates in Nasarawa State, Nigeria. J Infect Dev Ctries. 2009; 3(7): 539–547. PubMed Abstract | Publisher Full Text\n\nFeldblum PJ, Lie C, Weaver MA, et al.: Baseline factors associated with incident HIV and STI in four microbicide trials. Sex Transm Dis. 2010; 37(10): 594–601. PubMed Abstract | Publisher Full Text\n\nKalichman SC, Simbayi LC, Cain D, et al.: Heterosexual anal intercourse among community and clinical settings in Cape Town, South Africa. Sex Transm Infect. 2009; 85(6): 411–415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMwakagile D, Mmari E, Makwaya C, et al.: Sexual behaviour among youths at high risk for HIV-1 infection in Dar es Salaam, Tanzania. Sex Transm Infect. 2001; 77(4): 255–259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPriddy FH, Wakasiaka S, Hoang TD, et al.: Anal sex, vaginal practices, and HIV incidence in female sex workers in urban Kenya: implications for the development of intravaginal HIV prevention methods. AIDS Res Hum Retroviruses. 2011; 27(10): 1067–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrewer DD: Knowledge of blood-borne transmission risk is inversely associated with HIV infection in sub-Saharan Africa. J Infect Dev Ctries. 2011; 5(3): 182–198. PubMed Abstract | Publisher Full Text\n\nRamjee G, Gouws E, Andrews A, et al.: The acceptability of a vaginal microbicide among South African men. Int Fam Planning Perspect. 2001; 27(4): 164–170. Reference Source\n\nBrody S, Potterat JJ: Assessing the role of anal intercourse in the epidemiology of AIDS in Africa. Int J STD AIDS. 2003; 14(7): 431–436. PubMed Abstract | Publisher Full Text\n\nFerguson A, Morris C: Assessing the role of anal intercourse in the epidemiology of AIDS in Africa. Int J STD AIDS. 2003; 14(12): 856. PubMed Abstract | Publisher Full Text\n\nSimbayi LC, Kalichman SC, Jooste S, et al.: Risk factors for HIV-AIDS among youth in Cape Town, South Africa. AIDS Behav. 2005; 9(1): 53–61. PubMed Abstract | Publisher Full Text\n\nSchwandt M, Morris C, Ferguson A, et al.: Anal and dry sex in commercial sex work, and relation to risk for sexually transmitted infections and HIV in Meru, Kenya. Sex Transm Infect. 2006; 82(5): 392–396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGenberg BL, Kulich M, Kawichai S, et al.: HIV risk behaviors in sub-Saharan Africa and Northern Thailand: baseline behavioral data from Project Accept. J Acquir Immune Defic Syndr. 2008; 49(3): 309–319. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorhason-Bello IO, Oladokun A, Enakpene CA, et al.: Sexual behaviour of in-school adolescents in Ibadan, South-West Nigeria. Afr J Reprod Health. 2008; 12(2): 89–97. PubMed Abstract\n\nDeJanes W: Knowledge, substance use, and gender differences associated with HIV infection risk among youth in a South African township. Med Sociol Online. 2009; 4(2): 3–25. Reference Source\n\nKazaura MR, Masatu MC: Sexual practices among unmarried adolescents in Tanzania. BMC Public Health. 2009; 9: 373. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOla TM, Oludare BA: Sexual practices and knowledge about HIV/AIDS among Nigerian secondary school students. Int J Health Res. 2008; 1: 197–205. Reference Source\n\nStadler JJ, Delany S, Mntambo M, et al.: Sexual coercion and sexual desire: ambivalent meanings of heterosexual anal sex in Soweto, South Africa. AIDS Care. 2007; 19(10): 1189–1193. PubMed Abstract | Publisher Full Text\n\nKarim SS, Ramjee G: Anal sex and HIV transmission in women. Am J Public Health. 1998; 88(8): 1265–1266. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInstituto Nacional de Saúde (INS), Instituto Nacional de Estatística (INE), ICF Macro:Inquérito Nacional de Prevalência, Riscos Comportamentais e Informação sobre o HIV e SIDA em Moçambique 2009. INS, INE, and ICF Macro, Calverton, Maryland, USA 2010. Reference Source\n\nCenters for Disease Control and Prevention:Antenatal pre-test session flipchart.2011.\n\nMatambo R, Dauya E, Mutswanga J, et al.: Voluntary counseling and testing by nurse counselors: what is the role of routine repeated testing after a negative result? Clin Infect Dis. 2006; 42(4): 569–571. PubMed Abstract | Publisher Full Text\n\nInstituto Nacional de Estatística (INE), Ministério da Saúde (MS), ORC Macro: Moçambique Inquérito Demográfico e de Saúde 2003. INE, MS, and ORC Macro, Calverton, MD, USA 2005.\n\nAction Aid Mozambique, Save the Children UK, UNICEF: Stepping Stones Pilot Program Evaluation: Zambezia 19991999. Reference Source\n\nStepping Stones Evaluation - District of Mandimba.2002. Reference Source\n\nDepartment of Health (Republic of South Africa), South African Medical Research Council: South Africa Demographic and Health Survey 1998.2002. Reference Source\n\nNorthern Cape Department of Health, Academy for Educational Development/Capable Partners Programme South Africa: A rapid assessment of the extent to which knowledge, attitude and practice of maternal nutrition, infant, and young child feeding in the context of PMTCT of HIV are integrated in the community.2008.\n\nTuli K: Ndwedwe Child Survival Project KwaZulu Natal, South Africa.2006. Reference Source\n\nVersteeg M, Couper I, Mbiza E, et al.: Knowledge, attitudes and practices regarding HIV and AIDS in the Segwaelane and Damonsville communities. Madibeng Centre for Research, Brits, South Africa; 2005. Reference Source\n\nCrush J, Raimundo I, Simelane H, et al.: Migration-induced HIV and AIDS in rural Mozambique and Swaziland. Idasa, Cape Town; 2010. Reference Source\n\nPettifor AE, Rees HV, Steffenson A, et al.: HIV and sexual behaviour among young South Africans: a national survey of 15-24 year olds. Reproductive Health Research Unit, University of the Witwatersrand 2004. Reference Source\n\nBanana C: An investigation into the risk behaviour regarding HIV transmission among youth in Bulawayo. PhD thesis. University of South Africa 2007. Reference Source\n\nOunga T, Okinyi M, Onyuro S, et al.: Knowledge of HIV survival on skin-piercing instruments among young adults in Nyanza Province, Kenya. Int J STD AIDS. 2009; 20(2): 119–122. PubMed Abstract | Publisher Full Text\n\nGreen EC, Zokwe B, Dupree JD: The experience of an AIDS prevention program focused on South African traditional healers. Soc Sci Med. 1995; 40(4): 503–515. PubMed Abstract | Publisher Full Text\n\nSwarns RL: Mozambique enlists healers in AIDS prevention. New York Times, December 6, 1999. Reference Source\n\nMozambique News Agency: HIV/AIDS – new training manual for traditional healers. January 9, 2011. Reference Source\n\nAgência de Informação de Moçambique: HIV/SIDA: em preparação manual de treino para \"curandeiros\". January 8, 2011. Reference Source\n\nAssociacao dos Barbeiros, Mozambique Ministerio de Saude, Central de Educacao em Saude Publica: O barbeiro e o SIDA, por cada corte uma lamina nova, proteja a sua vida e a dos seus clientes [poster];1992.\n\nHainsworth G, Zilhão I, Badiani R, et al.: From inception to large scale: the Geração Biz Programme in Mozambique. World Health Organization, Geneva 2009. Reference Source\n\nHattori MK, Richter K, Greene J, et al.: Trust, caution, and condom use with regular partners: an evaluation of the trusted partner campaign targeting youth in four countries. Soc Marketing Q. 2010; 16(2): 18–48. Reference Source\n\nAllen S, Meinzen-Derr J, Kautzman M, et al.: Sexual behavior of HIV discordant couples after HIV counseling and testing. AIDS. 2003; 17(5): 733–740. PubMed Abstract | Publisher Full Text\n\nWawer MJ, Gray RH, Sewankambo NK, et al.: Rates of HIV-1 transmission per coital act, by stage of HIV-1 infection, in Rakai, Uganda. J Infect Dis. 2005; 191(9): 1403–1409. PubMed Abstract | Publisher Full Text\n\nKingsley C, Peters B, Babaahmady K, et al.: Heterosexual and homosexual partners practising unprotected sex may develop allogeneic immunity and to a lesser extent tolerance. PLoS ONE. 2009; 4(11): e7938. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeters B, Whittall T, Babaahmady K, et al.: Effect of heterosexual intercourse on mucosal alloimmunisation and resistance to HIV-1 infection. Lancet. 2004; 363(9408): 518–524. PubMed Abstract | Publisher Full Text\n\nBrewer DD, Rothenberg RB, Potterat JJ, et al.: HIV epidemiology in sub-Saharan Africa: rich in conjecture, poor in data. Int J STD AIDS. 2004; 15: 63–65. Publisher Full Text\n\nBrody S, Potterat JJ: Establishing valid AIDS monitoring and research in countries with generalized epidemics. Int J STD AIDS. 2004; 15(1): 1–6. PubMed Abstract | Publisher Full Text\n\nBrewer DD, Hagan H, Sullivan DG, et al.: Social structural and behavioral underpinnings of hyperendemic Hepatitis C virus transmission in drug injectors. J Infect Dis. 2006; 194(6): 764–772. PubMed Abstract | Publisher Full Text\n\nGisselquist D, Friedman E, Potterat JJ, et al.: Four policies to reduce HIV transmission through unsterile health care. Int J STD AIDS. 2003; 14(11): 717–722. PubMed Abstract | Publisher Full Text\n\nCorrea M, Gisselquist D, Gore DH, et al.: Blood-borne HIV: risks and prevention. Orient Longman Private Limited, Chennai. 2008. Reference Source\n\nGisselquist D, Correa M: Don’t get stuck with HIV: how to protect yourself during health care and cosmetic procedures. 2011. Reference Source"
}
|
[
{
"id": "50",
"date": "17 Jul 2012",
"name": "Suzanne Gaudreault",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article, and I think it makes a positive contribution to the evidence base. However, I think the introduction understates the level of knowledge that people with HIV are likely to have about HIV (see below).The discussion also neglects to consider the fact that due to stigmatization of this disease, many people with HIV do not admit that they have it or even that they have been tested for it. Unfortunately, this is hard to study or measure, but in practice, I have seen that it is especially true for men, even after they have known about their disease and have been on ART for a long time. In this study people who admitted to having been tested before were rightfully excluded from the analysis, but the discussion should acknowledge that stigma and other factors may mean that many people who actually knew about their status already (but lied about it) could still have been included in the data, seriously confounding the analysis.In countries like Mozambique which receive significant foreign assistance to deal with the HIV epidemic (PEPFAR, Global Fund or other) – people with HIV who are aware of their status typically receive much more intensive education about HIV (through treatment programs and social programs for people with HIV) than the general public. The content of this education is globally standardized enough that it typically places considerable emphasis on the modes of transmission of HIV. Also, as for people with any health condition, people with HIV are inherently more likely to pay attention to any information on HIV that they come across (public awareness campaigns, etc.).",
"responses": [
{
"c_id": "53",
"date": "19 Jul 2012",
"name": "Devon Brewer",
"role": "Author Response",
"response": "Thank you very much, Dr. Gaudreault, for your review.I agree that it is possible that some previously tested, infected persons might deny having tested previously and that they might have learned about blood exposures and anal sex as routes of transmission during HIV testing or clinical care. However, several lines of evidence suggest that such “knowledge through denied prior testing” is rare, and that even if this bias were present, it cannot account for the association between knowledge and HIV infection.In Class’ (2012) research, adult HIV patients and parents and other caretakers of HIV infected children in Maputo, Mozambique, responded to open-ended questions about routes of HIV transmission. Fifty-five percent (12/22) of the adult HIV patients and 62% (16/26) of parents and caretakers were aware of blood-borne modes of HIV transmission. All respondents aware of blood-borne HIV transmission reported learning of these modes of transmission exclusively from unofficial, non-clinical, non-public health sources, such as “on the street.” Class’ (2012) respondents reported that none of their clinical providers ever discussed blood-borne transmission as a potential explanation for how infection occurred.It is unclear the extent to which blood-borne HIV risks are covered in HIV counseling in southern Africa. As I demonstrated in this article and a prior report (reference 18), public HIV education campaigns in southern Africa have not in the past and do not currently address blood-borne HIV risks, even though they are often funded and guided by large international aid agencies such as USAID, the Global Fund, PEPFAR, DFID, and UNAIDS. Therefore, it is reasonable to question how well such risks are addressed in these countries’ HIV counseling programs.Any potential bias in reporting prior HIV testing by infected persons cannot account for the strong national-level inverse association between knowledge of blood-borne HIV risks and HIV infection (see reference 18). Countries in which many people are aware of such risks have much lower HIV prevalences than countries in which few people are aware of these risks.Also, potential bias in reporting prior HIV testing by infected persons is irrelevant to the association between uninfected mothers’ knowledge of blood-borne HIV risks and HIV infection in their children (reported in the current article). Children of uninfected mothers who know about these risks are much less likely to be infected than children of uninfected mothers who do not know about these risks.Furthermore, even if infected persons who denied prior HIV testing were included in the data I analyzed, their inclusion could not generate the observed inverse association between knowledge of blood-borne risks and HIV infection. If they had learned about blood-borne risks during testing or clinical care, and reported such awareness in the Mozambique AIS, their responses would produce a tendency toward a positive, rather than inverse, association between knowledge of blood-borne risks and HIV infection.ReferenceClass D. The socio-political economy of antiretroviral treatment as HIV prevention . Doctoral dissertation, University of Trento, 2012. Available at http://eprints-phd.biblio.unitn.it/803/."
}
]
},
{
"id": "100",
"date": "27 Jul 2012",
"name": "Nigel Livesley",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper has a number of serious flaws.a) The literature is quoted selectively and issues are oversimplified.The paper states that blood exposure is important but does not state that the preponderance of studies show that sexual exposure is by far the most common mode of HIV transmission.For example it doesn’t reference articles such as:\n\nUnsafe injections and transmission of HIV-1 in sub-Saharan Africa.\n\nThe global burden of disease attributable to contaminated injections given in health care settings.\n\nQuantifying HIV-1 transmission due to contaminated injections.The paper seems to claim that knowledge about blood borne infections account for the regional differences in HIV prevalence. A single citation (a paper by the same author) for this claim is provided. Other possibilities are not discussed such as:\n\nUnderstanding the differences between contrasting HIV epidemics in east and west Africa: results from a simulation model of the Four Cities Study\n\nEcological and individual level analysis of risk factors for HIV infection in four urban populations in sub-Saharan Africa with different levels of HIV infection.The paper also implies that condoms are not protective against HIV. Again this is not what the preponderance of studies shows:\n\nCondom effectiveness in reducing heterosexual HIV transmission.The author also notes the reference Allen S et al. (2003) as evidence for the lack of an association between condoms and HIV incidence but the study found that incident HIV infection was found in 5.4% of testing intervals when discordant couples had unprotected sex and in 2.6% of testing intervals when the discordant couples did not have unprotected sex.b) The interpretation of the results are not always supported by the data in the paper:The Author states:“After adjustments for demographic factors and sexual behavior variables, the measures of knowledge of blood-borne risks become inversely related to HIV infection. That is, respondents who reported that HIV can spread through shared needles/razors and blood contact were less likely to be infected than those who reported that HIV does not spread through such means, once demographics and sexual behaviors were held constant”.However, it is unclear how the author reaches this conclusion. The OR for the above relationships are about 0.9 and the confidence intervals are wide. This is not evidence of a relationship.The author also states:“The adjustments for potential confounders also caused the positive associations for knowledge of vaginal sex risk in women and knowledge of anal sex risk in men to disappear. However, the adjusted associations for knowledge of anal sex risk in women and knowledge of vaginal sex risk in men remained meaningfully positive.”It is again unclear what “meaningfully positive” means. The OR for women and anal sex has wide CI and doesn’t meet statistical significance. A definition of what the criteria for meaningfully positive are would be useful.And finally; The OR for men’s understanding of vaginal sex is close to being statistically significant.c) The paper’s conclusion is not supported:The author claims that:“HIV education campaigns in Mozambique and elsewhere in sub-Saharan Africa should include a focus on risks from blood exposures and anal sex.”However, the data in the paper doesn’t suggest that this is called for, and even if the data were supportive of the need to do this, there are opportunity costs associated with doing so. What should health workers or health educators not do to create time and money for this change in message?\n\nIs the title appropriate for the content of the article? Yes\n\nIs the abstract a suitable summary of the article? No (see above)\n\nIs the article well constructed and clear? No (see above)\n\nIs there adequate analysis, including information on how the data were analyzed (e.g. programs, code, stats etc.)? There is no information on how the author decided that some results were important\n\nAre the conclusions sensible and balanced? No (see above).\n\nHave any potential biases or competing interests been disclosed? Yes",
"responses": [
{
"c_id": "50",
"date": "30 Jul 2012",
"name": "Devon Brewer",
"role": "Author Response",
"response": "I thank Dr. Livesley for his review. He claims that I cited selectively from the literature and oversimplified issues, and that my interpretation of the results and the conclusions are not supported by the data. In this reply, I demonstrate that these charges are incorrect. Livesley states that “the preponderance of studies show that sexual exposure is by far the most common mode of HIV transmission” and criticizes me for not citing three articles that he thinks support this view. However, there is no solid empirical evidence that vaginal sex is the dominant mode of HIV transmission in sub-Saharan Africa, because studies with designs rigorous enough to determine modes of transmission have not yet been conducted. In the last paragraph of the current article, I wrote “to determine the modes of transmission of HIV in sub-Saharan Africa with confidence, researchers must assess blood and sexual exposures comprehensively in incident HIV cases and controls, trace their contacts corresponding to such exposures, and sequence the DNA in infected persons’ HIV isolates56 57 58.” My colleagues and I have published this plea dozens of times in the last nine years, and no other researchers have attempted to conduct the necessary research. Simply stating that vaginal sex is the dominant mode of transmission, or wishing it to be the case, does not make it so; rather, in science, strong empirical evidence must be presented to support a claim. My colleagues and I carried out a study in Calabar, Nigeria in which we assessed blood and sexual exposures fairly comprehensively in incident HIV cases and controls (Peters et al., 2009). We found that diverse blood exposures were associated with incident HIV infection. To my knowledge, no epidemiologic study of HIV infection before or since has matched or surpassed the strength of the research design of the Calabar study. Consequently, little can be concluded about the dominant modes of HIV transmission in Africa. Livesley cites Gisselquist D et al. (2004), Hauri et al. (2004), and White et al. (2007) as sources to support the hypothesis that vaginal sex is the dominant mode of HIV transmission in sub-Saharan Africa. These citations that do not provide support for this hypothesis. Gisselquist et al. assessed a small set of risk factors (and only one blood exposure, receipt of injections) in relation to incident HIV infection in Rwanda. As I have noted, it is not possible to determine modes of transmission with such a design, especially when blood exposures were so incompletely assessed. Hauri et al. and White et al. reported results from modeling exercises on the share of HIV infections that may be due to unhygienic medical injections (just one of many blood exposures encountered by Africans). I and others disagree with many of the assumptions and parameter estimates used and emphasized in their simulations (because they are at odds with empirical data). Their models also lack several key risk components of unhygienic injection practices. I refrain from a detailed technical critique of these studies here. Crucially, neither modeling exercise produced an estimate of the share of HIV transmission due to vaginal sex. Furthermore, modelers have not been able to simulate HIV epidemics that resemble African epidemics with parameter estimates derived from empirical data on Africans’ vaginal sex behavior (Brewer et al., 2007). Livesley faults me for citing my prior article that showed a strong inverse association between knowledge of blood-borne HIV risks and HIV prevalence at the national level in sub-Saharan Africa (Brewer, 2011). It is unclear why this is problematic, as the result is robust, and consistent with individual-level results and historical prevention campaign experience. Livesley notes that I did not discuss other possibilities for national variation in HIV prevalence. Such variation is not the focus of my current article; instead, I focused on knowledge of HIV transmission risks in Mozambique. Nonetheless, it is worth evaluating the two sources Livesley cites, both based on the Four Cities Study, for other explanations of variation in HIV prevalence across Africa. The Four Cities Study did not involve assessing participants’ blood exposures. Perhaps even more importantly, the results from the Four Cities Study showed that putative sexual risk factors (such as sex partner concurrency and other high risk sexual behaviors) were inversely associated (in the case of concurrency) or not associated with HIV prevalence in the four cities (Auvert et al., 2001; Lagarde et al. 2001; Rothenberg et al., 2002). In my F1000Research article, I stated that there is a “lack of association between condom use in vaginal sex and incident HIV infection14 52 53″ (p. 5) and cited three articles on studies conducted in sub-Saharan Africa (the only three I was aware of at the time that reported on the association or lack thereof). These three articles showed inconsistent results (Feldblum et al., 2010: positive association; Wawer et al., 2005: no apparent association; Allen et al., 2003: inverse association), which I summarized as a lack of association. Livesley cites Weller and Davis-Beaty 2007 for the hypothesis that condoms protect against HIV transmission during vaginal sex. However, Weller and Davis-Beaty’s summary included studies from regions outside of Africa, did not account for confounding of condom use with other risk factors (such as common blood exposures in Africa), and did not include Feldblum et al. and Wawer et al. results. About half of the studies in Weller and Davis-Beaty’s summary were based on condom use measures that applied to anal sex as well as vaginal sex. Consequently, Weller et al. results cannot be used as an estimate of the protective effect of condom use during vaginal sex in sub-Saharan Africa or elsewhere. The protective effect of condom use during anal sex, though, has been well-established in other research. Livesley questions my description of adjusted odds ratios of 0.81 to 0.95 for the association between knowledge of blood-borne HIV risks and prevalent HIV infection as indicating an inverse relationship. By definition, odds ratios under 1.0 reflect inverse or negative relationships. The adjusted odds ratios were in the inverse direction for two different measures of knowledge of blood-borne HIV risks and for both women and men. The confidence intervals for these odds ratios do include 1.0. However, the consistency of the association across measures and sample subgroups, and similarity in magnitude of the association to similar associations in neighboring and nearby countries mean that it is warranted to describe the relationship as inverse. Livesley criticizes me for characterizing the adjusted odds ratios between knowledge of anal sex and HIV infection in women (AOR = 1.18) and knowledge of vaginal sex and HIV infection in men (AOR = 1.85) as “meaningfully positive.” In this context, I intended “meaningfully positive” to refer to associations that were mild to moderate in magnitude. Livesley also correctly noted that “the OR for men’s understanding of vaginal sex is close to being statistically significant.” This result means that Mozambican men who believed that HIV can be transmitted through vaginal sex without a condom were twice as likely to be infected with HIV as men who did not believe vaginal sex was a risk. Livesley argues that the results in my article do not suggest that HIV education programs in sub-Saharan Africa should include a focus on blood-borne risks and anal sex. The results show that Mozambican adults who were unaware of blood-borne transmission were at increased risk for HIV infection, and that children of uninfected mothers who were unaware of blood-borne transmission were at much increased risk for HIV infection. Almost half of Mozambican adults did not know anal sex was an HIV transmission risk. In my opinion, these findings justify including a focus on blood-borne risks and anal sex in HIV education efforts. Livesley further wonders how such topics could be incorporated in HIV prevention and education programming. Successful examples abound. As I discussed in my 2011 article, there is a “heavy emphasis on avoiding blood-borne risks in HIV education programs for primary school students in rich countries [69,70], travelers to poor (especially African) countries [71-73], and employees of the United Nations working in poor countries [74,75]” (p. 194). Most of these educational efforts have been standard and ongoing for over two decades. The risk of anal sex has also been continuously emphasized in HIV prevention and education programs in rich countries. Moreover, during the last 30 years, there have been many instances of HIV education campaigns highlighting blood-borne risks in west and central Africa (Brewer, 2011), although it is uncertain how widespread and consistent these efforts have been. These examples show that people can be more fully informed of the range of HIV risks without additional program cost. The fundamental question is moral and ethical. On what grounds can HIV prevention and education programs in Africa, funded by taxpayers and sponsored by international agencies, foreign aid agencies, and national governments, continue to deprive people of highly relevant, life-saving information?"
}
]
},
{
"id": "101",
"date": "02 Aug 2012",
"name": "Carlos Morel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article. However, I am sure there are some sections where readers would like to have some more information e.g. how the adjustments for demographic and sexual behaviors were performed.",
"responses": [
{
"c_id": "52",
"date": "02 Aug 2012",
"name": "Devon Brewer",
"role": "Author Response",
"response": "I thank Dr. Morel for his review. I computed the adjusted odds ratios with multiple logistic regression. If there are any other points that require more information, I hope reviewers and readers will highlight those sections, so I can clarify them."
}
]
}
] | 1
|
https://f1000research.com/articles/1-1
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.