The SCF (Skp1p–cullin–F-box) ubiquitin ligases are a family of multi-subunit E3s that are responsible for ubiquitination of cell cycle regulators at the G1–S phase transition [2][7][8].

SCF complexes have common Skp1 and cullin subunits, but distinct F-box protein subunits that contain either a WD40 domain or a leucine-rich repeat domain that binds directly to phosphorylated SCF substrates [8][9][10].

In Saccharomyces cerevisiae, the G1-cyclins, Cln1p and Cln2p, bind SCFGrr1 [11][12], whereas the Cdc28p kinase inhibitor, Sic1p, has been shown to bind SCFCdc4 [13][14][15], where Grr1p and Cdc4p are the F-box components of these SCFs.


The anaphase-promoting complex (APC), or cyclosome, another multi-subunit E3, is responsible for the ubiquitination of cell regulators at the metaphase–anaphase and mitosis–G1 transitions [4].

The APC is comprised of a core complex of proteins and two WD40-containing proteins, Cdh1 (called Cdh1p or Hct1p in S. cerevisiae) and Cdc20, that bind to, and have been proposed to activate, the APC [16][17][18][19][20][21][22].

Cdc20 binds the APC (APCCdc20) during mitosis.

APCCdc20 is required for the degradation of the anaphase inhibitors known as the securins (Pds1p in S. cerevisiae and Cut2p in Schizosaccharomyces pombe), thereby triggering sister chromatid separation [23][24][25][26][27][28][29].

Cdh1 binds to the APC (APCCdh1) in late mitosis and G1 and is responsible for ubiquitination of the mitotic cyclins (Clb1–4p in S. cerevisiae) during these cell cycle phases [16][17][19][22][30].

APCCdh1 is also responsible for the degradation of Cdc20, thereby restricting APCCdc20 activity to a narrow window from G2 when Cdc20 is synthesized to late mitosis when Cdc20 is degraded by APCCdh1 [18][31][32].

In contrast, Cdh1 levels remain relatively constant during the cell cycle, but phosphorylation of Cdh1 by the mitotic kinase Cdc2 (Cdc28p in S. cerevisiae) blocks Cdh1 binding to the APC, thereby preventing APCCdh1 activity during S, G2, and M phases when Cdc2/Cdc28p activity is high [22][30][33][34][35].


Two degradation motifs have been identified in APC substrates.

The destruction box (D box), with the consensus sequence R-x-x-L-x-x-x-x-N/D/E, is important for the degradation of most APC substrates [36][4].

Recently, human Cdc20 (hCDC20), which lacks a D box, was found to contain a new degradation signal called a ‘KEN box’ with the consensus sequence K-E-N-x-x-x-D/N that was critical for its degradation by APCCdh1 [37].

Subsequently, a few other APC substrates containing both KEN box and D box motifs have been identified.

These APC substrates are human CDC6, a protein required for DNA replication, human securin, and the mitotic cyclin A in Drosophila melanogaster [38][39][29].

Functional KEN boxes in yeast proteins have not yet been reported.


Because direct interactions between Cdc20 or Cdh1 with APC substrates have not been demonstrated, generally these proteins are believed to activate the APC, possibly by inducing allosteric changes in core APC subunits leading to specific substrate binding and ubiquitination.

However, indirect evidence for substrate binding has led others to suggest, by analogy to the F-box proteins of the SCFs, that Cdc20 and Cdh1 may bind and target protein substrates to the APC [16][17][32][1][40][41].

We found previously that Hsl1p, a 170-kD budding yeast protein kinase that negatively regulates the Swe1p protein kinase in a bud morphogenesis checkpoint pathway [42][43][44][45], is degraded via the APC [1].

Like most APC-substrates, Hsl1p contains a D box that is important for its degradation.

Hsl1p associates with both Cdc20p and Cdh1p based on both two-hybrid assays and coimmunoprecipitation from yeast extracts, indicating that it might interact directly with these proteins [1].

Given these findings, we believed that Hsl1p might serve as a useful model substrate for understanding how proteins destined for degradation are recognized by the APC.


In this study we set out to understand which domains of Hsl1p are important for both recognition and turnover by the APC machinery.

We noticed that Hsl1p contains a potential KEN box and were interested in addressing the following questions using Hsl1p as a model APC substrate: 1) Do KEN boxes serve as degradation signals in yeast? 2) Can Hsl1p bind directly to Cdc20p and/or Cdh1p? 3) What is the role of the D box and/or KEN box in APC recognition?

We discuss a model for substrate recognition by the APC machinery.


The specificity of ubiquitin-mediated protein degradation with regards to the selection of substrates to be polyubiquitinated has only been determined rather recently.

Substrate targeting by the N-end rule and HECT (homology to E6AP carboxyl terminus) domain ubiquitin ligases occurs through substrate-specific binding domains.

In contrast, the SCF complex recruits substrates through a substrate adaptor protein, the F-box subunit.

Despite evidence showing that Cdc20 and Cdh1 bind and activate the anaphase-promoting complex (APC) in a substrate-specific manner, there is no evidence that the activating protein and substrate interact directly; hence, no clear model exists for the mechanism of APC activation or recruitment of substrates.

We show here that the activators Cdc20 and Cdh1 can associate with substrates via their N termini.

In the absence of APC, Cdc20 and Cdh1 bind substrates reflecting Cdc20–APC and Cdh1–APC specificity.

The N termini of Cdc20 and Cdh1 provide specificity functionally, as demonstrated by the generation of active chimeras that display the specificity corresponding to their N termini.

Thus, Cdc20 and Cdh1 act as both substrate recognition and activating modules for APC.


Ubiquitin-mediated protein degradation regulates important cellular processes from cell cycle progression to Wnt signaling [1][2][3].

Attachment of a polyubiquitin chain to a protein targets that protein for degradation by the 26S proteasome [4][5].

Ubiquitin becomes covalently attached to a substrate by interaction with a cascade of enzymes.

The last in the series, a ubiquitin protein ligase (E3), transfers the ubiquitin to a lysine on the substrate protein [6].

In the N-end rule pathway, extending from bacteria to humans, a destabilizing residue at the N terminus directs proteins for ubiquitination via the E3 N-recognin/Ubr1p (Saccharomyces cerevisiae) or E3α (in mammals).

Direct binding of substrates to N-recognin/E3α is observed [7][8][9].

Similarly, HECT domain E3s are highly conserved from yeast to vertebrates.

They share homology in their C termini to E6AP and contain a conserved C-terminal cysteine residue that forms a thioester with ubiquitin; they bind substrates via their N termini [10].

E6AP targets the src kinases [11], HHR23A and HHR23B [12].

The HECT domain RSP5 in yeast targets Gap1 and Fur4 by interaction with its N-terminal WW domains [13][14][15].

The fission yeast pub1 targets Cdc25 [16].

Xsmurf1 binds and targets Smad1 for degradation [17].

Two ubiquitin protein ligases play a role in the cell cycle: the SCF (Skp1/Cullin/F-box) complex and the anaphase-promoting complex (APC) [1][2][3].

Both complexes contain a protein with cullin homology (Cul1/Cdc53 in the SCF complex, APC2 in the APC) [18][19][20] and a RING-H2 finger protein, (Rbx1 in the SCF complex and APC11 in the APC) [19][21][22][23].

Recruitment of substrates to the SCF complex occurs via the F-box subunit.

F-box proteins interact with the Skp1 subunit of the SCF complex via the F-box motif [24].

F-box proteins fall into three categories, those with WD repeats (Fbw), leucine-rich repeats (Fbl), or other domains (Fbx) [3].

Binding to substrates requires substrate phosphorylation and is thought to occur in the non-F-box domains [3].

Substrate binding has been shown to occur for Cdc4 via its WD domain [25], for Grr1 via its leucine rich repeats [26][27], and for Skp2 via its other domain [28].

In some cases, however, the F-box protein may not be sufficient for substrate interaction; homo- or heterodimerization of the F-box or another factor may be required [3].

The APC contains several additional subunits without a defined role; substrate interaction could occur through one of these proteins.

APC activation requires its binding to the WD repeat containing proteins Cdc20 or Cdh1.

The Cdc20- and Cdh1-bound forms of the APC demonstrate different substrate specificities [29][30].

Cdc20–APC recognizes destruction box (D box) containing proteins [31][32][29], and Cdh1–APC recognizes proteins containing either a D box or a KEN box [30].

No direct binding of activating protein to substrate has been demonstrated.

In one case, the APC substrate Hsl1p, a Swe1p inhibitor, was identified through a two-hybrid interaction screen with Cdc20, but without addressing direct interaction [33].

In another case, the putative substrate cyclin D was shown to bind to other APC subunits by αCdc27 immuno-precipitation analysis [34].

More recently, the interaction between cyclin A–Cdk2 with the WD repeats of Cdc20 [35] or Cdh1 [36] has been reported for the recognition of Cdc20 and Cdh1 as substrates of cyclin A–Cdk2.

Recruitment of substrate to the APC might occur through activating protein, through binding to one of the other subunits of APC, or through a combination of both.

In this report we present evidence that Cdc20 and Cdh1, the substrate-specific adaptors of the APC, interact with substrates in an APC-independent manner.

We show that this interaction occurs through their N termini and does not require the WD repeats.

We show that this binding does not require APC and can occur directly between purified proteins.

Specificity is retained by the N terminus, as demonstrated by active chimeras between Cdc20 and Cdh1 when their N termini and WD repeats are swapped.

N-terminal fragments of the activators are potent APC inhibitors both in vitro and in vivo, and we introduce a binding assay that could be used to identify additional APC targets.


Brown dwarfs, gravitationally condensed objects whose masses are too low for equilibrium hydrogen burning, occupy the mass range between the lowest mass stars (∼ 0.07 M ⊙ ; Burrows et al. 1997) and the giant extrasolar planets (∼ 0.01 M ⊙ ; Marcy & Butler 1998).

The lowest mass stars and brown dwarfs only slightly cooler than M dwarfs are classified as spectral type "L" (Kirkpatrick et al. 1999, Martín et al. 1997) and have T ef f in the range ∼1500-2000 K. Deter- mining if L-type objects are brown dwarfs is difficult, because the luminosities and effective temperatures of brown dwarfs are a function of both age and mass (e.g. Burrows et al. 1997).


The first unambiguous brown dwarf, Gl 229B, was discovered as a compan- ion to a nearby M dwarf by Nakajima et al. ( 1995).

This object is cooler than any L dwarf and has CH 4 in its atmosphere implying T eff <1300 K (e.g. Fegley & Lodders 1996) and a sub-stellar nature.

The re- cent discoveries of methane dwarfs (tentatively given a "T" spectral clasification) in the field by Strauss et al. ( 1999), Burgasser et al. ( 1999, 2000), Cuby et al. ( 1999) and Tsvetanov et al. ( 2000) demonstrate that objects like Gl 229B can form singly.


A striking characteristic of known T dwarfs is the similarity of their spectra, which resemble those of the gaseous solar system planets and are very differ- ent from even the coolest L dwarfs.

The L dwarf spec- tra are characterized by the disappearance of the TiO and VO bands (which are strong in M dwarf spectra), the presence of atomic alkali lines and CO bands, and increasing depth of the H 2 O bands as T ef f decreases.

The T dwarfs are characterized by very deep H 2 O and CH 4 bands in the 1-3 µm region.

Alkali metal fea- tures are present between the bands, but the 2.3µm CO bands are absent, due to reduced CO abundance and the overwhelming strength of the CH 4 absorp- tion.


A large remaining gap in the stellar to gas giant planet spectral sequence lies between the previously observed types L and T. In this paper, we report pho- tometry and 0.8-2.5 µm spectroscopy of three very red, faint objects identified in the imaging data of the Sloan Digital Sky Survey (SDSS) and observed at the United Kingdom Infrared Telescope (UKIRT) and the Hobby-Eberly Telescope (HET).

These ob- jects have spectra intermediate between those of the previously known L and T dwarfs, showing the on- set of CH 4 absorption at 1.6 and 2.2 µm, while still retaining observable CO absorption at 2.3 µm.

We propose that they represent the warm end of the "T" spectral sequence, yet to be defined in detail.


The three objects described here were selected from SDSS photometric data.


SDSS photometry is ob- tained with a CCD camera at Apache Point Obser- vatory (APO) which images the sky almost simulta- neously in five filters: u ′ , g ′ , r ′ , i ′ , and z ′ (the data presented here use a preliminary calibration; while we denote the bands by u ′ etc, the magnitudes are de- noted by u * etc.).


The details of the data acquisition and the photometric and astrometric calibration are described by Gunn et al. ( 1998), York et al. ( 2000) and Lupton et al. ( 2000).


SDSS photometry is in the AB ν system (Fukugita et al. 1996) and the magni- tude scale is modified to deal with low signal-to-noise ratios (Lupton et al. 1999).


The L and T dwarfs identified in the SDSS (e.g. Strauss et al. 1999, Fan et al. 2000) are undetected in the u ′ and g ′ bands.

The L dwarfs have i * − z * > 1.6 while the T dwarfs have i * − z * > 3.

We prepared a candidate L and T dwarf list for follow-up obser- vations by searching the SDSS data for point sources with i * − z * > 1.6.


Because of their extremely red colors, some very red, faint objects are detected only in the z ′ band and a search for them can be contam- inated by defects.

Therefore, additional constraints were imposed: (1) all objects blended with neighbors, or affected by data defects, were removed; (2) an ob- ject was required to be detected at ≥3σ twice: in both i ′ and z ′ , in two observations of that region of sky, or detected in the 2MASS database; (3) a z ′ -band- only single detection was required to have z ′ <19.0.


The three objects discussed in this paper were selected from a total sky area of 225 deg 2 , giving an approxi- mate and very preliminary surface density similar to the 1 per 75 deg 2 for SDSS T dwarfs found by Tsve- tanov et al. ( 2000).

Table 1 gives, for the three new objects, the J2000 position (accurate to 0.

′′ 2) and AB magnitudes at i ′ and z ′ .

Table 1 also lists photometry obtained for the L dwarf SDSS 0539 (Fan et al. 2000).

Here- after we identify the objects by the first four digits of their Right Ascension.


SDSS 0837 was found in two SDSS runs, 1999 March 21 and 2000 February 8; the z * values agree to 0.1 mag and the positions to 0.

′′ 3.

SDSS 1021 is a z ′ -only detection in data from 2000 February 12 and is also in the 2MASS database.

The brightest object, SDSS 1254, has z * =18.0 and was found in data taken on 2000 February 2.

Finding charts for the new objects are shown in Figure 1.


J, H and K photometry was obtained using UKIRT's near-infrared camera, UFTI, for SDSS 0837 and SDSS 1254 on 2000 March 2 and for SDSS 0539 and SDSS 1021 on March 14.

Both nights were photometric with seeing ∼0.

′′ 8.

The results are given in Table 1 in the UKIRT (not AB) system - we give JHK on the UFTI system as well as colors on the established IRCAM system for comparison to earlier work.


Fig- ure 2 shows a composite color-color plot containing the data from Table 1 and, for comparison, a sample of late M and L dwarfs as well as the SDSS T dwarfs SDSS 1624 and SDSS 1346 (Strauss et al. 1999; Tsve- tanov et al. 2000), whose spectra closely resemble that of Gl 229B.

Lower limits are shown for i * − z * for the z ′ -only detections.

The late M and L dwarfs show a steady progression towards redder J−K and i * − z * colors with later spectral type, however the T dwarfs have redder i * −z * colors and bluer J−K colors due to the strong absorption by CH 4 in the H and K bands.

The J−K colors of the new objects lie between those of the previously known L and T dwarfs.


A red spectrum of SDSS 1254 was obtained on 2000 February 29 with the Low Resolution Spectrograph (LRS; Hill et al. 1998a,b, Schneider et al. 2000) at the prime focus of the of the HET (Ramsey et al. 1998).

The spectrum covers the wavelength range 5100-9800Å at a resolution of ∼ 20Å, but no flux is detected below ∼6500Å.


Spectra of the objects in Table 1 were obtained in the J, H and K bands at UKIRT on 2000 February 28 - March 1 and March 13-15 using the facility spectrograph CGS4 (Mountain et al. 1990) at R∼400.

Integration times were 30-60 minutes per band.

Tel- luric absorption was removed and the broad-band spectral shapes corrected by comparison with spectra of bright F stars (with photospheric lines removed) taken immediately before and/or after each observa- tion.

The spectra in each band were scaled to match the UKIRT magnitudes using the UFTI filter profiles.

UKIRT z-band spectra were also obtained for SDSS 1254 and SDSS 0539.

For SDSS 1254 the HET spec- trum is used for λ < 0.84µm, and for SDSS 0539 the red APO spectrum (Fan et al. 2000) is used for λ < 0.89µm.


The final flux-calibrated spectra are shown in Fig- ures 3 and 4.

Figure 3 shows the spectrum of SDSS 1254 split into four panels, with the main absorption features marked.

Figure 4 compares the spectra of the new objects with those of the L5 dwarf SDSS 0539 (Fan et al. 2000) and the T dwarf SDSS 1624 (Strauss et al. 1999).


The spectra show: increasingly red col- ors at λ < 1 µm, probably due to pressure broadened Na I and K I absorption (Burrows et al. 2000) com- bined with decreasing T ef f ; increasing absorption in the H 2 O bands at 1.15 µm, 1.4 µm and 1.9 µm; in- creasing absorption at 1.6-1.7 µm and longward of 2.2 µm by CH 4 combination and overtone bands; de- creasing absorption in the CO 2.3µm band.

In par- ticular, the absorption maxima of the CH 4 2ν 2 +ν 3 , 2ν 2 , and ν 2 +ν 3 bands at 1.63, 1.67, and 2.20 µm be- come progressively deeper.

The CO 2-0 bandhead at 2.294 µm is seen in the new objects, but the 3-1 band- head at 2.323 µm is absent; it is considerably weaker at these temperatures and also is overwhelmed by the strong CH 4 ν 3 + ν 4 absorption at 2.315 µm.


The spectra in Figure 4 reveal a clear spectral sequence.

Thus SDSS 0837, SDSS 1254 and SDSS 1021 are examples of the sought after "L/T transi- tion" objects.

However, as suggested by Figure 4, they might more properly be regarded as examples of early T dwarfs, where the "T" spectral type is de- fined by the presence of CH 4 absorption in the H and K bands.

The strongest short wavelength infrared band of CH 4 is the ν 2 band, centered at 3.3 µm, and objects in which this band is present but the shorter wavelength bands reported here are absent must ex- ist over a narrow temperature range.


However, mea- surements at 3.3 µm are considerably more difficult from the ground than are H and K band measure- ments, and objects which show only the 3.3 µm CH 4 band perhaps should be classified as L rather than T (Kirkpatrick et al. ( 1999) suggest that the T class be defined by the presence of CH 4 absorption in the K band).

As the three objects presented here are at the CO/CH 4 transition temperature they are likely to have T ef f ≈ 1300 K (Fegley & Lodders 1996) and therefore masses of 20-70 M Jupiter for ages in the range 0.3-5 Gyr (Burrows et al. 1997).

Figure 2 shows that SDSS easily identifies T dwarfs by their extremely red i * − z * color, including the early T dwarfs identified in the present paper.


On the other hand, the J−K colors are similar to those of the common M dwarfs and these early T objects are thus very difficult to select on the basis of the near- infrared colors alone (although the later T dwarfs are blue and can be selected this way).

Many more L and T dwarfs will be found in the SDSS imaging data.


The SDSS is a joint project of the University of Chicago, Fermilab, the Institute for Advanced Study, the Japan Par- ticipation Group, Johns Hopkins University, Max-Planck- Institute for Astronomy, Princeton University, United States Naval Observatory, and the University of Washington.

The site, Apache Point Observatory, is operated by the Astrophys- ical Research Consortium.

Funding has been provided by the Alfred P. Sloan Foundation, the member institutions, NASA, NSF, the U.S. DoE, and Monbusho Japan.

The SDSS Web site is at http://www.sdss.org/.


UKIRT is operated by the Joint Astronomy Centre on behalf of the UK Particle Physics and Astronomy Research Council.

We thank Tim Carroll for his expert and cheerful help with the UKIRT observations, and M.-C. Liang for assistance during the 28 Feb - March 1 run.


The Hobby-Eberly Telescope is a joint project of the Univer- sity of Texas at Austin, Pennsylvania State University, Stan- ford University, Ludwig-Maximillians-Universität München, and Georg-August-Universität Göttingen.

The HET princi- pal benefactors are William P. Hobby and Robert E. Eberly.

We thank Phillip MacQueen, Grant Hill, Matthew Sheltrone, and Marsha Wolf for help with the HET data.


XF and MAS ac- knowledge support from the Research Corporation, NSF grant AST96-16901, the Princeton University Research Board, and a Porter O. Jacobus Fellowship.

GRK is grateful for support from Princeton University and from NASA via grant NAG- 6734.

DPS thanks the NSF for support via grant AST99-00703.


We present near-infrared spectra for a sample of T dwarfs, including 11 new discoveries made using the 2 Micron All Sky Survey.

These objects are distinguished from warmer (L-type) brown dwarfs by the presence of methane absorption bands in the 1–2.5 km spectral region.

A first attempt at a near-infrared classification scheme for T dwarfs is made, based on the strengths of bands and the shapes of the 1.25, 1.6, and 2.1 km flux peaks.

Subtypes T1 V–T8 V are defined, and spectral indices useful for classification are presented.


The subclasses appear to follow a decreasing scale, based on bands and the properties of L and T dwarfs with known distances.

However, we speculate that this scale is not linear with spectral type for cool dwarfs, due to the settling of dust layers below the photosphere and subsequent rapid evolution of spectral morphology around T K. Similarities in near-infrared colors and continuity of spectral features suggest that the gap between the latest L dwarfs and earliest T dwarfs has been nearly bridged.

This argument is strengthened by the possible role of as a minor absorber, shaping the K-band spectra of the latest L dwarfs.


Finally, we discuss one peculiar T dwarf, 2MASS 0937–2931, which has very blue near-infrared colors due to suppression of the 2.1 km peak.

The feature is likely caused by enhanced collision-induced absorption in a high-pressure or low-metallicity photosphere.


We present 0.6–2.5 µm, R >∼ 400 spectra of twenty-seven cool, low luminosity stars and substellar objects.

Based on these and previously published spectra we develop a preliminary spectral classification system for L and T dwarfs.

For late L and T types the classification system is based entirely on four spectral indices in the 1-2.5 µm interval.

Two of these indices are derived from water absorption bands at 1.15 µm and 1.4 µm, the latter of which shows a smooth increase in depth through the L and T sequences and can be used to classify both spectral types.

The other two indices make use of methane absorption features in the H and K bands, with the K band index also applicable to mid to late L dwarfs.

Continuum indices shortward of 1 µm used by previous authors to classify L dwarfs are found to be useful only through mid L subclasses.

We employ the 1.5 µm water index and the 2.2 µm methane index to complete the L classification through L9.5 and to link the new system with a modified version of the 2MASS “Color-d” index.

By correlating the depths of the methane and water absorption features, we establish a T spectral sequence from types T0 to T8, based on all four indices, which is a smooth continuation of the L sequence.

We reclassify two 2MASS L8 dwarfs as L9 and L9.5 and identify one SDSS object as L9.

In the proposed system methane absorption appears in the K band approximately at L8, two subclasses earlier than its appearance in the H band.

The L and T spectral classes are distinguished by the absence and presence, respectively, of H band methane absorption.


In the last six years, brown dwarfs, objects of mass too low to sustain equilibrium hydrogen burning, have gone from being a theoretical concept with no observed examples to being observationally ubiquitous [1].

Young brown dwarfs have been discovered in large numbers in star-forming regions [2][3][4][5].

Objects with masses ranging from just substellar to those of the Solar System’s giant planets have been found directly or indirectly as companions to nearby stars [6][7][8][9][10][11][12].

In addition, large numbers of isolated brown dwarfs have been found in the solar neighborhood [13][14][15].

These observations have led to controversy regarding the distinction between brown dwarfs and planets, with some suggesting that the lower mass limit for brown dwarfs be defined by the ability to burn deuterium (mass greater than ∼13 MJ, [16]), and others preferring the definition that a brown dwarf is any substellar object that forms via the same process as a star (i.e. not from a protoplanetary disk).

Brown dwarfs are of interest in their own right. They have compact structures, low temperatures, and atmospheric constituents that are markedly different from low mass stars.

The lowest mass brown dwarfs more closely resemble the gas giant planets than stars, and as such provide insight into the physical and chemical properties of extrasolar giant planets that are at present inaccessible for direct study.

After an initial period of deuterium-burning and, for the more massive brown dwarfs, hydrogen-burning, there is no internal energy source and a brown dwarf evolves without reaching thermal equilibrium.

This leads to age- and mass-dependent spectral types, in contrast to main sequence stars, and indeed eventually to effective temperatures lower than that of any stellar photosphere.

It is generally agreed that the very coolest stars and the warmer brown dwarfs (1300 K <∼ Tef f <∼ 2200 K) require a new spectral class, commonly known as ‘L’ [17][18] and currently known cooler brown dwarfs (Tef f <∼ 1300 K), require the addition of an additional class, ‘T’ [18].

The L dwarf sequence is characterized by the disappearance of the red TiO and VO bands from the optical (0.6–1.0 µm) spectrum, the increasing dominance at those wavelengths by broad absorption resonance lines of Na I and K I, and strong H2O absorption bands and persistent CO overtone bands in the 1-2.5 µm region [19][18][20][21].

In T dwarfs the CO bands are replaced by stronger and more extensive absorptions of CH4 in the H and K bands, and there is further strengthening of the water bands [8][22][23][24][25][26].


The range of L spectral types has been characterized using optical spectroscopy of objects from the Deep Near-Infrared Survey, DENIS [27] and the Two Micron All Sky Survey, 2MASS [28], while first attempts to describe the range of the ‘T’ spectral sequence are given by [29][30][31], whose sample is drawn from the imaging data of the Sloan Digital Sky Survey, SDSS [32].

The SDSS imaging data allow the discovery of T dwarfs covering a fairly wide range of spectral characteristics.

In pure infrared surveys such as 2MASS, the degeneracy of the colors of T dwarfs with those of stars (see [31]) permits only the latest T dwarfs to be selected readily (without the use of optical photometry).

This paper presents optical (0.6–1.0 µm) and infrared (1.0–2.5 µm) R >∼ 400 spectroscopy of new L and T dwarfs selected from the SDSS together with new observations of some previously known M, L, and T dwarfs.

Following the discovery of transition objects with properties between those of the then known L and T classes [31], our goal has been to obtain sufficient spectra of L and T types over the entire 0.6-2.5 µm range and at sufficiently high resolution to allow the continuous characterization of the L and T dwarf spectral sequences.

Until now the L sequence has been described only on the basis of optical spectra, and hence it has not been not clear if its full extent has been observed.

In addition there are significant differences in the assignments of L subclasses for the latest objects [18][19].

Using new and previously published spectra, we have devised a set of infrared flux indices from which we have defined a complete L spectral sequence, a T spectral sequence extending to the coolest known brown dwarfs, and a distinct boundary between the L and T sequences.

One of the new infrared indices provides a firm link between our L sequence and those proposed by [19] and [18] on the basis of optical spectral indices.

The L and T sequences we have created are purely empirical and the ranges of spectral indices describing them are defined so that they progress smoothly through the known L and T dwarfs.

We have made no attempt to model the spectra or to derive physical quantities such as temperature or mass.

Some observational gaps remain in the T sequence, and it is quite likely that the sequence will eventually extend to one or more later subclasses as lower temperature objects are found.


The next two sections describe the selection of objects and the observations.

In Section 4 we discuss various indices for spectral typing and present the classification system.

Section 5 discusses the results, with emphasis on the definition of the L//T boundary and the link between infrared and optical classification of L dwarfs.

The conclusions are given in Section 6.

In an adjoining paper, [30] present an independently derived spectral sequence for T dwarfs, which bears many similarities to the one proposed herein.

In a companion paper by [21] we provide ZJHKL′M′ photometry of many of objects presented herein and we discuss the behavior of broad-band colors as a function of spectral class.


Nine morbidly obese (BMI=46.2± 7.7 kg/m 2 [mean±SD] and fat-free mass [FFM]=84.6± 16.6 kg) type 2 diabetic patients and six sex- and age- matched healthy volunteers (BMI=24.4±1.1 kg/m 2 ) were studied.

The patients were all characterised as having type 2 diabetes according to the criteria established by the American Diabetes Association (ADA) [24].

HbA 1c was 6.0-9.5%.


All individuals were randomly assigned to the 24 h metabolic study or to the OGTT either at baseline and 7-9 days after bariatric surgery.

The two tests were performed at an interval of 24-48 h.


At the time of the baseline study, all individuals were on a diet with the following average composition: 60% carbo- hydrate, 30% fat and 10% protein (≥1 g/kg body weight).

This dietary regimen was maintained for 1 week before the study.

All patients received the same parenteral nutrition regimen (about 7,100 kJ/day) during the first 6 days after surgery, then they were on free diet.

For the basal study, each individual spent 24 h (starting at 08:00 hours) on the metabolic ward.

Four meals were administered in the calorimetric chamber for a total energy intake of 104.5 kJ (25 kcal)/kg FFM: 16.4% at breakfast at 09:00 hours, 36% at lunch between 12:00 and 13:00 hours, 13.4% at an afternoon snack at 16:00 hours and 34.2% at dinner at 20:00 hours.

Diet composition was 16.7% protein, 9.8% fat and 73.5% carbohydrate at breakfast; 18.8% protein, 51.2% fat and 30% carbohydrate at lunch; 13.4% protein, 27.7% fat and 58.9% carbohydrate at snack time; and 18.9% protein, 49.8% fat and 31.3% carbohydrate at dinner.

The average diet composition was 16.9% protein, 34.6% fat and 48.5% carbohydrate.

Hourly blood samples were drawn from a central venous catheter for the measurement of glucose, C-peptide and NEFA concentrations.


OGTT A standard 75 g OGTT was performed in each patient at baseline and after surgery as well as in each volunteer, with blood sampling at 0, 30, 60, 90, 120, 150 and 180 min.

Insulin sensitivity was computed by the OGTT minimal model [25].


Body composition was evalu- ated, on a separate day, by the determination of total body water (TBW) using 0.19 Bq 3 H 2 O in 5 ml of saline administered as an intravenous bolus injection [26].

Blood samples were drawn before and 3 h after the injection.

Radioactivity was determined in duplicate on 0.5 ml of plasma in a beta scintillation counter (Model 1600TR; Canberra-Packard, Meriden, CT, USA).

Corrections were Diabetologia (2008) 51:1276-1284 1277 made for non-aqueous hydrogen exchange [27].

Water density at body temperature was assumed to be 0.99371 kg/l.

TBW (kg) was computed as 3 H 2 O dilution space (litres)× 0.95×0.99371.

FFM was obtained by dividing TBW by 0.732 [28].

Fat mass was obtained as the difference between body weight and FFM.


All procedures and measurements described for the basal study were repeated in the obese individuals at the follow-up.


This malabsorptive surgical procedure [29] consists of an approximately 60% distal gastric resection with stapled closure of the duodenal stump.

The residual volume of the stomach is about 300 ml.

The small bowel is transected at 2.5 m from the ileo-caecal valve, and its distal end is anastomosed to the remaining stomach.

The proximal end of the ileum, comprising the remaining small bowel (involved in carrying bilio-pancreatic juice but excluded from food transit), is anastomosed in an end-to- side fashion to the bowel, 50 cm proximal to the ileo- caecal valve.

Consequently, the total length of absorbing bowel is reduced to 250 cm, the final 50 cm of which, the so-called common channel, represents the site where ingested food and bilio-pancreatic juices mix.


The study was approved by the Institutional Ethics Committee of the Catholic University of Rome.

The nature and purpose of the study were carefully explained to all indi- viduals before they provided their written consent to participate.


Plasma glucose was measured by the glucose oxidase technique on a Beckman Glucose Analyzer (Beckman, Fullerton, CA, USA).

C-peptide was assayed by RIA (MYRIA; Technogenetics, Milan, Italy).

Serum NEFA was measured spectrophotometrically.


The 24 h ISR was estimated by using a previously proposed mathematical model of glucose-NEFA comodulation [19].

Model equations are reported and briefly explained in the appendix.

The model allows the computation of the ISR from the time-course of glucose and NEFA plasma concentrations.

To analyse the C-peptide data, the ISR model was complemented by the equations for the whole body C-peptide kinetics.

We have used the validated two- compartment model [30, 31], calculating the standard parameters of C-peptide kinetics for each individual as proposed by van Cauter et al. [ 31].


The ISR model parameters were estimated for each individual by weighted least-squares fitting of plasma C-peptide concentration data [19].

The least-squares index was minimised by means of a constrained Levenberg- Marquardt routine of the MATLAB library and the SDs of the estimates of individual parameters were evaluated by the Jackknife method [19].

From the parameter esti- mates, the 24 h individual ISR profile was derived.

For comparison, the ISR profile was also reconstructed from the plasma C-peptide concentration by the deconvolution method, according to van Cauter et al. [ 31].

The β-oxidation rate was computed for each individual by means of Eq. 3 in the appendix.


All data are expressed as means±SE, unless otherwise specified.

The Wilcoxon paired-sample test and the ANOVA test for repeated measurements, followed by a Tukey test were used for intragroup and intergroup comparisons, respectively.

Two-sided p<0.05 was considered significant.


The duration (ranging from 6 months to 10 years before the operation) and the degree of compensation of diabetes (HbA 1c ) were used as covariates in a multiple regression analysis where the dependent variable was the change in beta cell glucose sensitivity after BPD.


A non-significant weight loss (6.7±3.3 kg [mean±SD]) was observed 1 week after BPD.

The FFM after BPD was 80.9± 15.7 kg and was not significantly different from FFM before BPD.

The 24 h average concentrations of glucose before and after BPD (13.6±5.6 vs 7.8±2.6 mmol/l) were significantly different (p<0.01), and the glucose concentration after BPD was not significantly different from that found in controls (5.2±1.0 mmol/l).

The 24 h average concentration of C-peptide after BPD (1.2±0.7 nmol/l) was significantly (p<0.05) lower than that before BPD (1.7±0.9 nmol/l) and did not differ significantly from the average concentration in controls (0.7±0.3 nmol/l).

The 24 h average concen- trations of NEFA before and after BPD (0.64±0.08 vs 0.65±0.30 mmol/l) were not significantly different and were larger than in controls (0.31±0.08 mmol/l, p<0.05).

The time-course of NEFA plasma level over 24 h before and after BPD is reported in Fig. 1.


The estimates of model parameters are reported in Table 1.

For all individuals, the SD of the individual parameter estimates was consistently <20%.

The parameter p 1 , which represents the 24 h average beta cell sensitivity to both glucose and NEFA, was doubled 1 week following BPD (p<0.01).

Nevertheless, this parameter did not reach the values observed in healthy controls.


The parameter p 2 , which is related to the rate of long-chain acyl-CoA (LC- CoA) use in the cytosol, recovered completely after BPD, being substantially equal to the values found in controls.

Also p 3 , which expresses the efficiency of LC-CoA transport into the mitochondria, where LC-CoAs are 1278 Diabetologia (2008) 51:1276-1284 β-oxidised, reverted to normality.

Thus the ratio p 3 /p 2 , which represents the balance between LC-CoA β-oxidation and its use in the cytosol [19], was fully normalised early after the operation.


The integral over 24 h of the insulin secretion rate (ISR; cumulative insulin secretion, AUC ISR ) decreased from 277.1±144.4 to 198.0±107.6 nmol/m 2 (p< 0.02) and did not differ from that found in controls (135.0± 30.0 nmol/m 2 ).

The amplitude δ and the initial phase ϕ of the ISR circadian rhythm did not change significantly after BPD.

Whereas the amplitude was significantly higher in controls (p<0.001), the phase was not statistically different from before/after BPD.


The rate of β-oxidation in the beta cell was found to be completely normalised after the operation, from 0.032±0.012×10 −12 to 0.103±0.031× 10 −12 mmol/min per cell, having assumed a beta cell volume of 1.376×10 −12 l [32].

For the controls we found 0.098±0.052×10 −12 mmol/min per cell.


Figure 2a-c shows the model fitting of the 24 h C-peptide plasma concentration before and after the opera- tion in a representative diabetic patient.

The time-course of the ISR, estimated both by the model and by the deconvo- lution method, is also reported.

The cumulative 24 h insulin secretion attained the same value when computed by the mathematical model or by the deconvolution.

The peaks corresponding to the meals were reduced after the operation, shifting towards the values observed in controls (Fig. 2d,e).


The relationship between the static component of the ISR (denoted as S, see Eq. 1 in Appendix) and NEFA concentration is shown in Fig. 3a for controls and type 2 diabetic patients before and after BPD.

The curves are plotted at the glucose level (G) indicated, using the parameters p 1 , p 2 and p 3 of Table 1.

The points mark the 24 h average NEFA/ISR value with the average glucose concentration found in each group of individuals.

The variability of the curve with glucose level is shown only for controls.


Figure 3b shows the rate of β-oxidation per cell (B) as a function of plasma glucose concentration, computed by the model (Eq. 3 in appendix) in diabetic patients before and after BPD and in controls.

At least to our knowledge, no data are available in the literature on fatty acid oxidation in islets from obese diabetic humans or in healthy individuals.

Therefore we plotted in Fig. 3b, as a reference, the values of palmitate oxidation in Zucker lean rat islets, as deduced by the data reported in Nolan et al. [ 23] assuming that the average beta cellular protein content is 200 fg.


The time-courses of glucose and insulin concentration during the OGTT of diabetic patients and controls are shown in Fig. 4.

Both the fasting glucose and insulin plasma levels and their values at 120 min were dramatically reduced after BPD.

The ADA criteria to define diabetes mellitus (fasting plasma glucose ≥7 mmol/l or an OGTT with the 2 h postload value ≥11.1 mmol/l) were no longer fulfilled after the operation, as shown in Fig. 4, so the


The major results of the present study may be stated as follows:


In diabetic patients who underwent malabsorptive bariatric surgery, the beta cell glucose/NEFA sensitivity was doubled early after the operation, before a significant weight loss was achieved.


The shorter the time interval between diagnosis of type 2 diabetes and bariatric operation and the lower the severity of diabetes, the better was the recovery of beta cell function.


Fatty acid oxidation in the beta cell, as calculated by the mathematical model, was fully normalised after BPD, suggesting that the impairment of pancreatic fatty acid β-oxidation is a reversible phenomenon.


4. Insulin secretion was reduced mainly as a consequence of the normalisation of insulin sensitivity.

Circulating NEFA was indeed slightly, although not significantly, increased after BPD, probably as a consequence of the increased fat mobilisation by hormone-sensitive lipase, which is activated when the circulating insulin level drops.

The lack of a significant increase in NEFA concentration may be attributable to a higher insulin sensitivity of adipose tissue, which attenuates the potential rise in NEFA.


Points 1-4 are discussed in detail in the following.


One of the first pieces of evidence of diabetes resolution after bariatric surgery was reported by Pories et al. [ 33].

Although the surgical technique used in that study was the gastric bypass, whereas BPD was used in the present study, in our series also diabetes was reversed early after the operation when body weight was quite unchanged.


Insulin sensitivity exerts a modulating effect on the beta cell function, so that beta cells are initially able to compensate for insulin resistance, thus maintaining glucose homeostasis.

In fact, insulin sensitivity and beta cell function are inversely related, whereby the product of these two parameters is a constant referred to as the disposition index [34].

But insulin hypersecretion is no longer able to fully compensate for increasing insulin resistance when beta cell function is impaired, so that type 2 diabetes mellitus develops [35-40].

Individuals with impaired glucose tolerance and overt type 2 diabetes are character- ised by moderate to severe insulin resistance [40, 41].

For instance, Haffner et al. [ 42] reported that insulin resistance was present in >85% of a cohort of diabetic individuals.

It is interesting to note that Ferrannini et al. [ 41] have shown that a halving of beta cell glucose sensitivity or a halving of insulin sensitivity predicts a similar rise in the 2 h glycaemia after glucose challenge as well as in the fasting plasma glucose levels, suggesting that either the beta cell glucose sensitivity or the insulin resistance can equally contribute to hyperglycaemia in type 2 diabetic patients.


In our series, a reduction of insulin secretion was found after BPD in all individuals whereas the insulin sensitivity was significantly increased, suggesting that the improvement of insulin sensitivity may induce a corresponding improvement in beta cell glucose/NEFA sensitivity.


The effect of bariatric surgery in terms of recovery of beta cell function (Δp 1 ) was maximal in those individuals whose diabetes was diagnosed shortly before the operation, while the effect decreased linearly with the increase of diabetes duration.

The UKPDS data [43] clearly indicate that there is a progressive deterioration of pancreatic beta cell function as type 2 diabetes progresses.

In fact, type 2 diabetic patients at diagnosis had about 50% reduction in pancreatic beta cell function and were insulin resistant, whereas over time there was a reduction (about 4%/year) in the beta cell function regardless of treatment, without change in insulin resistance.


In agreement with our results Schauer et al. [ 44] found, in a cohort of 1,160 patients who underwent laparoscopic Roux-en-Y gastric bypass, that the factors that negatively correlated with type 2 diabetes mellitus resolution included the preoperative duration of diabetes, high levels of HbA 1c and insulin therapy, which are all measures of diabetes severity.

In contrast, the weight loss was positively associated with diabetes resolution.

Our data are not affected by the weight loss variable, since our patients were studied very early after the operation when no significant weight loss was attained.


On the basis of the present observations, it may be hypothesised that in patients with long-standing type 2 diabetes the defects in beta cell mass predominate, whereas the patients with a short duration of diabetes primarily exhibit an impairment in beta cell function.


Beta cell fatty acid oxidation A β-oxidation impairment in the beta cells has been described by Frigerio et al. [ 45].

The anti-epileptic drug topiramate exerts a protective effect against INS-1E beta cell lipotoxicity induced by prolonged exposure to high levels of fatty acids, by enhancing the cellular capacity of lipid clearance.

In fact, this drug enhances the β-oxidation rate, suggesting that a protective mechanism against lipotoxicity could be the inhibition of lipid accumulation in the cytoplasm through increased fatty acid oxidation.

Furthermore, fa/fa rat islets, a useful replica of the human phenotype of adipogenic type 2 diabetes, exhibit a markedly increased lipogenic capacity and a decreased oxidative capacity [46]; unused NEFA in islets are esterified and over time an excessive quantity of triacygly- cerol is deposited [46].


Our observation that fatty acid oxidation rate in beta cells is normalised a few days after BPD suggests that the impairment of fatty acid oxidation is reversible and that it is not related to the fatty acid overloading of beta cells.

In fact, circulating NEFA levels were not statisti- cally changed from before BPD, when their oxidation rate was calculated to be three times lower than in healthy controls.


At least to our knowledge this is the first evidence of a defect in beta cell fatty acid oxidation in type 2 diabetes in humans.

This defect is completely reversed shortly after BPD even before complete normalisation of beta cell glucose sensitivity.

It is likely that this prompt reversibility might depend on the removal of some competitive inhibitory action exerted on carnitine palmitoyltransferase 1 (CPT-1).


It has been reported that in type 2 diabetic patients the insulin pulses related to meals are decreased in amplitude and disorganised in their temporal pattern [47, 48] and that, in most diabetic patients, these abnormal patterns did not improve with weight loss [49].


In our post-BPD patients, although insulin secretion was reduced, it was still sufficient to provide a normalisation of the insulin-dependent glucose disposal.

This effect occurred in spite of the non-significant reduction in circulating NEFA levels, suggesting that other mechanisms should intervene.

In particular we hypothesise that insulin sensi- tivity normalisation may be the first to occur and that, secondarily, the need for insulin secretion diminishes.


Malabsorptive bariatric surgery in type 2 diabetic patients restores β-oxidation in pancreatic beta cells, doubles the glucose-NEFA sensitivity and reverts to normality the diabetic condition.

Since circulating NEFA levels (which together with glucose concentration determine the ISR) do not decrease significantly, it is likely that it is the normal- isation of insulin sensitivity that causes the reduction of insulin secretion.

A fivefold increase of insulin sensitivity, as was observed in our study population, is much higher than that usually obtained by insulin sensitising agents, and it is independent of weight changes.

Therefore, it is conceivable that the altered intestinal physiology produced by the surgical operation could be the causative factor of these findings.

In particular, the exclusion of part of the small bowel from food transit might act to avoid local production of factor(s) worsening insulin sensitivity.


We stress that the results of the present study cannot be extended to all type 2 diabetic patients since they derive from a specific category of patients where the insulin secretion is still high enough in spite of frank diabetes mellitus.


The authors declare that there is no duality of interest associated with this manuscript.


We describe here in a synthetic form the mathematical model of NEFA-glucose comodulation of beta cell insulin secretion [19].


As in other mathematical models of glucose-stimulated insulin secretion [20, 50], the ISR is given by the sum of a static and a dynamic component.

The static component is modelled here according to the view that a dual signalling pathway that involves both glucose and LC-CoA regulates the stimulation of insulin secretion [22] (see Fig. 5).

The kinetics of LC-CoA formation from exogenous NEFA is represented by a single rate constant k. Cytosolic LC-CoA has two possible fates: bind to CPT-1 and be transported into mitochondria to undergo β-oxidation, or be enrolled in diacylglycerol (DAG) synthesis and enhance the ISR, possibly via activation of protein kinase C. A key aspect of the model is that it describes in a very simplified way the inhibitory action of glucose (via malonyl-CoA) on the binding of LC-CoA to CPT-1 and thus on the rate of LC- CoA transport into mitochondria.

The following equation for the static ISR component S (expressed in nmol/min) has been obtained [19]:


where G and F are the glucose and NEFA concentration respectively, the coefficient p 1 is the beta cell sensitivity to both glucose and NEFA, p 2 is related to the rate of LC-CoA use in the cytosol, and p 3 expresses the efficiency of LC- CoA transport into mitochondria where LC-CoAs are β-oxidised.

The term in square brackets in Eq. 1 is a sort of 'potentiation factor' that accounts for the action of fatty acids on insulin secretion and the influence of glucose on fatty acid metabolism.

By adding the dynamic component to the static component, we obtain the expression for the ISR:


Because the parameter p 1 was found to change with the time during the 24 h meal test [19], we have added to p 1 a circadian oscillation [20].

Thus the glucose/NEFA sensitiv- ity coefficient is expressed as p 1 +δ sin(2πt/T+ϕ), where p 1 is the average value over the 24 h, δ is the amplitude of the oscillation, T is the period equal to 24 h, and ϕ is the phase at the initial time (t=0 taken at 08:00 hours).

The ISR model has the following unknown parameters: k, p 1 , p 2 , p 3 , δ, ϕ, k d , to be estimated from the data.


In the hypothesis that the LC-CoA transported into the mitochondria is completely β-oxidised, the rate of β-oxidation B (expressed as mmol/min per cell) can be determined as the difference between the NEFA influx into the beta cell and the rate of LC-CoA use in the cytosol.

This leads to the expression:


where V is the volume of the beta cell.

It may be seen that B decreases with the increase of the glucose concen- tration G.


To the Editor: In a recent paper, Salinari and colleagues reported normalisation of glucose metabolism associated with improved pancreatic beta cell sensitivity to nutrients derived from a mixed meal, 1 week after malabsorptive bariatric surgery (bilio-pancreatic diversion), in morbidly obese patients with type 2 diabetes [1].

This was observed before the patients had lost a significant amount of weight.

On the basis of timed serum insulin, C-peptide, NEFA and plasma glucose measurements, they calculated the effects of NEFA and glucose on the insulin secretion rate (ISR).

The plasma glucose level was decreased to within the normal range after the surgery, but the serum NEFA level was unchanged.

Using a mathematical model, the authors concluded that the improved ISR after bariatric surgery was due to the restoration of normal NEFA oxidation in the beta cell, which enhanced ISR despite the significant decrease in the plasma glucose level.

This study is of great interest to us because we consider the malonyl-CoA pathway and the resultant NEFA augmentation of insulin exocytosis to be vital for glucose-stimulated insulin secretion (GSIS), especially for the replenishment of the readily releasable pool of insulin granules during second-phase secretion [2, 3].

Prior to the visual demonstration and molecular elucidation of intracellular granule trafficking, this process was known conceptually as ‘potentiation’ [4], ‘augmentation’ [5] or ‘amplification’ [6].


The paper makes no mention of factors other than glucose and NEFA that are also involved in meal-induced insulin secretion, such as incretins [7], parasympathetic input and amino acids, to name but a few.

Of note, an increase in the serum level of the incretin glucagon-like peptide 1 (GLP-1) is documented 1 week after bilio-pancreatic diversion, and this is thought to be responsible for long-term effective weight reduction through satiety [7].

GLP-1 acts on the beta cells to increase the level of cyclic AMP, leading to replenishment of the readily releasable pool of insulin granules during GSIS.

This is an ATP-sensitive K+ channel-independent insulinotropic action [8].

Interestingly, NEFA also augments GSIS via a mechanism that does not involve this channel [5].

Salinari et al. use a mathematical model of glucose–NEFA comodulation to estimate the ISR [1].

Therefore, given the lowered basal glucose, lowered glucose excursion after the meal, unchanged NEFA and increased ISR, they will naturally conclude that the effect of NEFA has been enhanced/amplified.

However, in our opinion, the increased beta cell potentiation [8], achieved through the GLP-1–cyclic AMP pathway after surgery, is expected in their patients, in line with previous findings in patients who underwent the same operation, at the same postoperative time-point [7].

A different conclusion might have been reached if the incretin effect had been taken into account.

The inclusion of the positive effect of the incretin pathway would have at least reduced, and at most cancelled out, the calculated effect of restored β-oxidation on ISR after surgery [1].


In conclusion, by not considering factors other than glucose and NEFA in meal-induced insulin secretion, Salinari et al. [ 1] may have overestimated the effect of restored NEFA oxidation on ISR after bariatric surgery.

The data itself are very important and have considerably expanded our understanding of the physiology of bariatric surgery.

However, a more comprehensive analysis of the ISR, one that at least takes account of the incretin effect, would be desirable.


To the Editor: In response to the letter of Komatsu and Aizawa [1] concerning our recent paper in Diabetologia [2], we wish to make the following points.

Because of space constraints we were not able to clarify the roles of the different parameters included in our model in detail.

Although, as noted by Komatsu and Aizawa, glucose and NEFA are the only inputs used to obtain the insulin secretion rate (ISR), the model response is also determined by the values of the parameters p1, p2 and p3.

These parameters are estimated by fitting the model to the C-peptide experimental data.

The equation for estimating ISR contains a function that includes both glucose and NEFA levels.

This function contains the parameters p2 and p3, which are related to beta cell fatty acid oxidation.

The parameter p1 acts as a multiplier of this function, and represents a further factor involved in the potentiation of the ISR.

Incretin action, in particular, is implicitly accounted for by the parameter p1 of the model.

The value of p1 in a given experimental setting is thus related to factors other than NEFA and glucose that determine the ISR.


We have found that the parameters p2 and p3 markedly change after bariatric surgery (BPD), becoming very close to control values (Table 1).

It therefore appears that defective β-oxidation is reversed after BPD.

However, we do not imply that this is the major factor responsible for the improvement in ISR.

In fact, after BPD, the NEFA level is unchanged whereas the glucose concentration is decreased, probably because of the increase in insulin sensitivity.

Parameter p1 is doubled after BPD, but does not reach the value found in controls.

We interpret this fact as evidence that an incretin-dependent increase in beta cell sensitivity to glucose–NEFA has occurred.

The incretin effect implicitly resides in the value of parameter p1.

Figure 3a of our paper [2] shows the effects of BPD on the model response.


In a previous article we applied this model to data from healthy volunteers during a hyperglycaemic clamp after either water or butter ingestion [3].

After the water load, the parameter p1 was half the average value observed using the 24 h multiple-meal test.

Butter alone was able to double parameter p1, possibly via the incretin effect.

In fact, in this paper we suggested that the potentiation of insulin secretion mediated by gastrointestinal hormones does not occur after water ingestion but can take place after a butter test meal [3].

We appreciate the suggestion made by Komatsu and Aizawa [1] regarding the effect of NEFA on the replenishment of the readily releasable pool of insulin granules during the second phase of insulin secretion.

We recently proposed a mathematical model of insulin granule trafficking in the beta cell [4] in which the dynamics of the various granule pools, including reserve, docked and primed, immediately releasable and fused granules, are represented.

Unfortunately, the paper by Aizawa and Komatsu [5] escaped our attention.

Extension of the model to include the effect of NEFA would be of great interest.


Successful infection control is essential in the treatment of chronic osteomyelitis and infected non-union.

Adequate sur- gical debridement remains key to achieving this goal (Fer- guson et al., 2017; Lew and Waldvogel, 2004).

Surgical debridement should be accompanied by systemic antibi- otic treatment.

Problems include low antibiotic concentra- tions due to inadequate perfusion of the bone and the sur- rounding soft tissues (van Vugt et al., 2016; Romano et al., 2014; Geurts et al., 2011) as well as the development of an- tibiotic resistance and the formation of biofilms (van Ges- tel et al., 2015; Lindfors et al., 2017).


High concentrations can be achieved with local treatment, e.g., with gentamicin- containing polymethylmethacrylate (PMMA) beads.

Cur- rently, the gold standard in treating chronic osteomyelitis is a two-stage procedure that involves the use of antibiotic- containing PMMA beads in the first procedural stage (Geurts et al., 2011; Lindfors et al., 2017; van Vugt et al., 2019).


However, the PMMA beads must be removed in a further operation (Ferguson et al., 2017; Geurts et al., 2011), and, after releasing the antibiotics, they can themselves act as a E. Steinhausen et al.: Bioactive glass vs. autologous bone graft foreign body, thereby creating a base for bacterial biofilms (Romano et al., 2014; Lindfors et al., 2017; Ferguson et al., 2014; Rahaman et al., 2014).


Bone reconstruction is necessary after successful infection control.

Autologous bone graft is the gold standard and has osteogenic, osteoinductive, and osteoconductive properties (Ferguson et al., 2017; Calori et al., 2011; De Long et al., 2007; Egol et al., 2015).

However, the volume that can be achieved is limited, and donor site morbidity is considerable (Pape et al., 2010).


Various bone substitutes have been developed in recent years to complement or even replace autologous bone graft- ing (Egol et al., 2015; Fillingham and Jacobs, 2016; Kurien et al., 2013).

An ideal bone substitute should be osteoconduc- tive, osteoinductive, biodegradable, and biocompatible (van Vugt et al., 2016; Calori et al., 2011; Fillingham and Ja- cobs, 2016).


Bone substitutes loaded with antibiotics have been developed for the treatment of bone infections.

They usually contain gentamicin (Romano et al., 2014; McNally et al., 2016; Fleiter et al., 2014; Lalidou et al., 2014), or al- ternatively tobramycin (Ferguson et al., 2014; McKee et al., 2010) or vancomycin (Luo et al., 2016), and make it possible to achieve high local antibiotic concentrations with low sys- temic levels without incurring the disadvantages of PMMA beads (Ferguson et al., 2017; Lindfors et al., 2017; Ferguson et al., 2014; McNally et al., 2016; Fleiter et al., 2014; McKee et al., 2010; Luo et al., 2016).


Clinical evidence in this area is still limited, however (Ferguson et al., 2017; Geurts et al., 2011).


Bioactive glass S53P4 is an inorganic bone substitute with antibacterial, osteoconductive, osteostimulative, and angio- genic properties (Cunha et al., 2018; Coraca-Huber et al., 2014).

The results of existing clinical studies of bioactive glass are promising (van Gestel et al., 2015; Lindfors et al., 2017; Ferrando et al., 2017; Lindfors et al., 2010; Auregan and Begue, 2015).


Here, we compare the outcome of bioactive glass S53P4 versus autologous bone grafts for filling defects in patients with chronic osteomyelitis and infected non-union.


Bioactive glass (BAG) (Bioglass S53P4; BonAlive ® Bio- materials Ltd., Turku, Finland) is an inorganic bone sub- stitute containing SiO 2 , Na 2 O, CaO, and P 2 O 5 .

Following implantation, sodium and basic ions are released, causing a rapid increase in pH and osmotic pressure.

A silicon dioxide- rich layer is formed on the surface.

Calcium and phosphate groups migrate to the surface to form a hydroxyl-apatite scaf- fold.

Osteoblasts migrate into this scaffold, proliferate, dif- ferentiate, and stimulate osteogenesis.

These surface reac- tions not only stimulate the formation of new bone, but also have an antibacterial effect (Lindfors et al., 2017; Coraca- Huber et al., 2014; Lindfors et al., 2010).


In addition, in vitro studies have shown an angiogenic effect with increased secretion of the vascular endothelial growth factor (VEGF) (Detsch et al., 2014).


We conducted a retrospective analysis of patients treated in our institution for chronic osteomyelitis and infected non- union who underwent filling of a bone defect with either autologous bone graft or BAG between July 2013 and Jan- uary 2017.


Inclusion criteria were age > 18 years, chronic os- teomyelitis and infected non-union (positive bacteriology and/or positive histology), filling of the bone defect with ei- ther autologous bone graft or BAG, and minimum follow-up of 12 months.

Exclusion criteria were aseptic non-unions and lack of follow-up (Fig. 1).


Descriptive epidemiologic data and details of the pre-, peri-, and post-operative courses were compiled from the digital patient files and processed anonymously.

Follow-up was analyzed up to a January 2018 cut-off.


The primary endpoint was successful control of infec- tion during follow-up (no clinical, laboratory or radiological signs of infection, closed soft tissues).

Secondary endpoints were bone healing and functional outcome focusing on mo- bilization with full weight bearing.

Any complications were recorded.


Bone healing was quantified by evaluating X-ray or CT imaging.

Healing was defined as the presence of complete cortical bridging in three out of four cortices or with no evi- dence of the fracture line.


Multi-resistant pathogens were defined as pathogens re- sistant to most antibiotics or antibiotic groups (methicillin- resistant Staphylococcus aureus, multi-resistant coagulase- negative Staphylococci, multidrug-resistant Gram-negative bacteria and extended spectrum ß-lactamase bacteria).


All patients were treated exclusively by the specialized team of the septic surgery department.

Decisions regarding treat- ment method were taken individually for each patient based on surgeon preference.


In all cases, radical debridement was carried out prior to filling the defect.

The surgeons took care to completely fill the defect and to press the implanted BAG into the surround- ing bone.

New samples were taken intraoperatively from all patients.


All patients received antibiotic prophylaxis periopera- tively for previously detected pathogens.

If an unknown pathogen was present, the patients received cefazoline as the standard antibiotic used in our institution.

Patients with an uncomplicated course and sterile samples did not re- ceive postoperative antibiotic treatment.

Patients with a pro- J. Bone Joint Infect., 6, 73-83, 2021 https://doi.org/10.5194/jbji-6-73-2021 longed course with multiple previous revisions or with per- sistent detection of pathogens received postoperative antibi- otic treatment.

Antibiotic treatment was chosen according to the pathogen and the antibiotic resistance pattern.

Antibiotics were given intravenously for 3 weeks and orally for an addi- tional 3 weeks.


Treatment groups were compared using Fisher's exact test for binary data and the Mann-Whitney U test for continuous data.

A p value < 0.05 was considered statistically signifi- cant.

Given the sample size of about 80 cases, the power of this study is limited, especially for even counts.

Significant differences for a theoretical prevalence rate of 20 % could be expected only if the prevalence was < 5 % or > 40 % in the other group.

For continuous measures, the detectable differ- ence was about half a standard deviation.

Statistical analysis was performed using SPSS (Version 24, IBM Inc., Armonk, NY, USA).

Multivariate analyses were conducted for com- plications, recurrence of infection, and osseous fusion.


Fifty-one patients received bioactive glass S53P4 (BAG), 32 patients autologous bone graft (AB), and 12 patients a com- bination of bioactive glass and autologous bone graft.

The outcomes for these 12 patients are described separately at the end of this section but are not analyzed statistically.

There- fore, the following results represent a total of 83 patients (Fig. 1).


The autologous bone graft and the bioactive glass group were comparable with respect to sex, localization and kind of fracture, and the number of previous operations and flaps (Table 1).

However, the patients of the BAG group were significantly older and had undergone an unsuccess- ful prior attempt at defect filling significantly more often than those in the autologous bone graft group.

Staphylococ- cus aureus and coagulase-negative Staphylococci were the most common pathogens involved in both groups.

Gram- positive pathogens were found significantly more often in the bioactive glass group.

Gram-negative pathogens were de- tected in both groups without significant difference.

Multiple pathogens and multidrug-resistant pathogens also did not dif- fer significantly between the groups (Tables 1, 2).


In the BAG group, an average of 11 cm 3 bioactive glass was implanted (minimum 5 cm 3 ; maximum 30 cm 3 ).

Intraopera- tive samples showed persistent bacteria in 17 patients of the BAG group and in 11 patients of the AB group, indicating no significant difference between the groups.

However, pa- tients of the autologous bone graft group had undergone a change in pathogens significantly more often (BAG 3.9 %; AB 28.1 %; p = 0.001).

Forty patients of the BAG group and 29 patients of the AB group received postoperative antibiotic treatment owing to a prolonged course or persistent detection of pathogens.

No significant differences were found between the two groups regarding postoperative antibiotic treatment (Table 3).


Patients of the autologous bone graft group had significantly longer follow-up than patients of the bioactive glass group (p < 0.001).


Overall, major and minor complications were found in 22 patients of the BAG group and in 20 patients of the AB group, indicating no significant difference between the groups.

Re- currence of infection occurred in 15 patients of the BAG group and in 6 patients of the AB group.

Statistical analy- sis also failed to show a significant difference between the groups with regard to recurrence of infection.

Two patients (6.3 %) suffered complications requiring revision surgery af- ter removal of autologous bone from the iliac crest.

No com- plications associated with bioactive glass were observed.


Forty-seven patients of the BAG group and 31 patients of the AB group achieved full weight bearing during follow- up, indicating no significant difference in this respect.

How- ever, patients of the BAG group achieved full weight bear- ing significantly more rapidly (BAG 5.9 ± 4.1 months, me- dian 6 months; AB 10.7 ± 8.6 months, median 10 months; p = 0.018).


The results for bone healing were similar in both groups, with bone healing being seen in 39 patients of the BAG group and in 25 patients of the AB group by the end of follow- up.

Again, patients in the BAG group accomplished bone healing more rapidly (BAG 9.5 ± 7.0 months, AB 10.8 ± 9.0 months).

Eleven patients of the AB group underwent another procedure to fill the defect, significantly more frequently than in the BAG group (BAG n = 8; p = 0.049).

In addi- tion, patients of the autologous bone graft group underwent reoperations significantly more frequently (BAG n = 24; AB n = 24; p = 0.014).

The rate of below-knee amputation due to recurrent infection during follow-up was comparable be- tween the two groups (BAG n = 3; AB n = 2; p = 1.00) (Ta- ble 3).


Detection of multi-resistant pathogens was associated with a significantly higher rate of incomplete osseous fusion (p = 0.033) and was also associated with a 3-fold increase in the risk of complications (odds ratio 3.091, 95 %-confidence in- terval 1.128-8.470).

Male patients had a significantly greater risk of recurrent infection.

Finally, there was a 3.5-fold in- creased risk of recurrent infection after local or free tissue transfer (odds ratio 3.508, 95 %-confidence interval 1.155- 10.660).


Twelve patients had a defect filled with a combination of BAG and autologous bone graft.

Intraoperative samples re- vealed persistent bacteria in two patients (16.7 %).

Full weight bearing was reached after 4.8 months, on average.

Nine patients (75 %) achieved complete bone healing dur- ing follow-up after an average of 7.6 months.

Four patients (33.3 %) developed complications.

Only one patient (8.3 %) had recurrent infection during follow-up.


The goals of osteomyelitis therapy are successful control of infection and the reconstruction of the bone.

Bioactive glass S53P4 is an inorganic bone substitute with antibacterial, osteoconductive, and osteostimulative properties (Coraca- Huber et al., 2014; Heikkila et al., 1995).


Our comparison of the outcomes of patients with chronic osteomyelitis and infected non-union who had a defect filled with either autologous bone graft or BAG revealed no sig- nificant differences between the groups with respect to re- currence of infection, bone healing, full weight bearing, or complications in general.


Some studies have investigated the use of BAG in patients with chronic osteomyelitis.

However, the number of cases in these studies was usually small, and most had no control group (Romano et al., 2014; Lindfors et al., 2017, 2010; Au- regan and Begue, 2015; McAndrew et al., 2013; Drago et al., 2013; Malat et al., 2018; Oosthuysen et al., 2020).

Ex- isting comparative studies compare BAG either with other bone substitutes (Table 4) or with PMMA beads (the gold standard for a two-stage procedure) and with the objective of investigating successful control of infection and dead space management of bone cavities, but not bone healing.


To our knowledge, ours is the first study to compare BAG with au- https://doi.org/10.5194/jbji-6-73-2021 J. Bone Joint Infect., 6, 73-83, 2021 tologous bone graft in patients with an infected non-union that includes fracture healing as a secondary endpoint.


The reinfection rate of patients with chronic osteomyelitis is reportedly as high as 21.2 % (Walenkamp et al., 1998), and the overall reinfection rate after implantation of BAG is 0 %- 14 % according to the current literature (Romano et al., 2014; van Gestel et al., 2015; Lindfors et al., 2017, 2010; McAn- drew et al., 2013; Drago et al., 2013; Malat et al., 2018; Tan- war and Ferreira, 2020).

We saw higher reinfections rates, especially in the BAG group.

However, the number of pa- tients treated with BAG in published studies is usually small.

Moreover, the definition of reinfection or persistence of in- fection used by other authors is less strict than ours.

Romano et al. ( 2014) defined a "fair" outcome as a wound with pro- longed drainage or serum leakage of up to 6 weeks and a "poor" outcome as no wound healing for more than 6 weeks or one requiring surgical intervention.


Lindfors et al. ( 2017) defined the outcomes of their patients in a similar way.

These findings would be interpreted as persistent infection in our assessment.

These differences in definition may explain the increased infection rate in our BAG group.


On the other hand, the infection rate of our autologous bone graft group cor- relates with that reported in other studies.

Staphylococcus aureus and coagulase-negative Staphylococci were the most common pathogens involved in both groups.

The presence of multi-resistant pathogens was associated with an increased risk of complications regardless of treatment.

These results agree with those in other publications.

An additional risk factor for recurrent infection in both groups was a previous local or free tissue transfer, as also reported by Lindfors et al. ( 2017).


All factors considered, we could not establish an advan- tage of BAG compared to AB with regard to infection con- trol.

There were no significant differences between our two groups in other respects; that is, we found no clear disadvan- tage for BAG.


Our rate of complete osseous fusion is 77.1 % without signif- icant difference between the groups at latest follow-up.


Pub- lished studies do not report comparable rates (Romano et al., 2014; van Gestel et al., 2015; Lindfors et al., 2017; Ferrando et al., 2017; Lindfors et al., 2010; McAndrew et al., 2013; Drago et al., 2013).


This is unsurprising because these stud- ies investigated the use of BAG only as a bone void filler in chronic osteomyelitis.


However, they describe progressive incorporation of BAG, and radiographs therein show partial incorporation at latest follow-up, although the biomaterial re- mained visible (Romano et al., 2014).


In addition, they de- scribe the formation of a thickened cortex (van Gestel et al., 2015).


BAG was not fully incorporated in our patients, ei- ther, and we observed the phenomenon of a thickened cortex (Fig. 2).


Ninety-four percent of our patients reached full weight bearing.

Thus, we assume that, although osseous fusion re- mained incomplete on some radiographs, bone consolidation was adequate to ensure stability.

Basically, these patients re- gained a working extremity.


We observed high complication rates in both groups.

The ac- tual rate of reinfection was higher in the BAG group, but patients of the AB group needed significantly more reoper- ations and significantly more additional defect fillings.


Our rate of complications from harvesting autologous bone grafts from the iliac crest is comparable with that reported in other studies (Dimitriou et al., 2011).

However, only complications requiring revision surgery are mentioned in our study.

Pain after removal of autologous bone graft from the iliac crest and the resulting delay in mobilization are not recorded, al- though nearly all patients suffer these complications.

Last but not least, harvesting an autologous bone graft from the ante- rior or posterior iliac crest requires prolonged surgery.


These drawbacks do not apply to the use of bone substitutes.

In our study, bioactive glass was not associated with adverse effects.

This matches the experience in the other published studies.

So far, there has been no report of BAG-associated compli- cations or formation of bacterial resistance (Romano et al., 2014; van Gestel et al., 2015; Drago et al., 2013).

The toler- ability of BAG is even described as superior when compared to other bone substitutes (Ferguson et al., 2014; McNally et al., 2016).

Van Gestel et al. ( 2015) referred to bioactive glass S53P4 as a potential new gold standard.


Lindfors et al. ( 2017) analyzed the largest series of patients (n = 116) with chronic osteomyelitis to date, but without a control group.

BAG was used in a one-stage procedure in most of the cases, with excellent results.

The authors high- light the possibility of one-stage use due to the antibacte- rial effect as the key advantage of BAG.

Other authors agree and underscore its angiogenetic and antibacterial effects (van Gestel et al., 2015; McAndrew et al., 2013; Drago et al., 2013), although radical debridement remains indispensable (Lew and Waldvogel, 2004; Lindfors et al., 2017; Simpson et al., 2001).

Geurts et al. ( 2011) compared various antibiotic-loaded bone graft substitutes in active or suspected infection with PMMA beads.

The authors argue that PMMA beads remain the gold standard even if they require a two-stage procedure.


Hence, new biomaterials show great potential, but the level of available evidence is still limited.

In contrast to autologous bone graft, BAG has antibacterial and angiogenetic effects.

These effects are especially advan- tageous in often poorly vascularized bone.

Previous studies show that bacterial biofilms can cause the infecting organism to be resistant to systemic antibiotic concentrations of up to 1000 times greater than normal therapeutic levels (McKee et al., 2010).

Such concentrations cannot be achieved with sys- temic application.


Recently, Ferrando et al. ( 2017) compared bioactive glass S53P4 (n = 12) with calcium sulfate antibiotic beads (n = 13).

They did not find significant differences with regard to complication rates and recurrence of infection, but the num- ber of patients was small.

The authors conclude that BAG without adjunctive use of local antibiotics is as effective as calcium sulfate antibiotic beads.


Van Vugt et al. ( 2016) evaluated the use of various bone graft substitutes (Osteoset T, BAG, PerOssal, Herafill-G) in a systematic review (n = 15 studies).

In general, they criti- cize weak study design, small number of patients, low levels of evidence (LoE 2b-3b), and a high risk of bias.

Interest- ingly, the reinfection rate (primary outcome) was higher in high-quality studies.

No significant differences were found between the different bone graft substitutes.

The authors con- clude that the results are inconclusive.


To our knowledge, no study to date compares or proves the superiority of BAG over autologous bone graft with respect to its antibacterial effect.


We combined bioactive glass S53P4 with autologous bone graft in treating large bone defects (n = 12) (Fig. 3).

The cur- rent literature contains no comparable in vivo studies.

An in vitro study has analyzed and verified the antibacterial activ- ity of BAG as a bone graft extender in combination with AB (Bortolin et al., 2018).

However, to date there is no evidence to indicate which mixing ratio of bioactive glass and autolo- gous bone graft is optimal.

The optimal technique - in layers versus mixed - also remains to be determined.

Yet our results in a small number of cases are excellent with respect to our primary and secondary endpoints.

Therefore, we prefer the combination of BAG and AB for the treatment of large bone defects.

However, further evidence in favor of this approach is needed.


Our study has some limitations.

First, it is a retrospective observational study - although with a control group.

The groups are of different sizes, and patients in the BAG group are significantly older.

The level of evidence is weak.

The maximum follow-up differs between the groups, with signif-


To our knowledge, this is the first study to compare the use of BAG and AB in patients with chronic osteomyeli- tis and infected non-union.

The results are promising.

BAG seems to be an appropriate bone substitute, not only for fill- ing bone defects in patients with chronic osteomyelitis, but also for achieving fracture healing in cases of infected non- union.

In our study, BAG was neither superior nor inferior to autologous bone graft with regard to our primary and sec- ondary endpoints.

Prospective randomized studies - as rec- ommended by many authors - would be desirable but are not truly feasible.

An analysis with matched pairs may be an al- ternative.


I read with interest the recent paper by Steinhausen et al. ( 2021) [1].

This paper reports the outcome of a retrospective review of bioactive glass compared to autologous bone graft (ABG) in a staged surgical protocol for treating established bone infection.

The authors claim that bioglass is equally effective as ABG in eradicating infection but acknowledge that their evidence is weak.


The surgical treatment of chronic bone infection has been reported for well over a century.

In 1931, Jacob Kulowski published his classical series of 130 cases of osteomyelitis, treated by Orr’s method (Kulowski, 1931) [2].

His surgical debridement technique was similar to that described by Steinhausen et al. ( 2021) [1], and he achieved a recurrence rate of 24% (mean follow-up: 19 months), in the pre-antibiotic era, with no defect fillers.

In this new series of 83 patients, the recurrence rate with bioglass was 29% and 19% for ABG.

Clearly the addition of bioglass or bone graft has not improved outcome.


The authors state that the longer follow-up period for the ABG group is not a source of bias because “most complications occurred within 12 months”.

This same group previously published 50 of their 51 bioglass cases (Malat et al., 2018) [3] with a mean follow-up of 12.3 months and a recurrence rate of 14%.

This has increased to 29% with a longer follow-up (mean 20.5 months).

It would appear that the recurrence rate doubled in the 8 months after the first year.


There are no large randomized trials directly comparing the use of bioglass with other options, and the authors correctly report that outcomes of small studies are inconclusive.

However, there are two high-quality animal studies (Xie et al., 2009; Boot et al., 2020) [4][5].

In a rabbit model of MRSA osteomyelitis, Xie et al. ( 2009) [4] reported that bioglass was no more effective than debridement alone (success rate 36% for debridement and 18% for debridement and bioglass) but the addition of local antibiotics to calcium sulfate or bioglass increased success to 73% and 81% respectively.

Boot et al. ( 2020) [5] studied the effect of hydrogel and bioglass in an infected tibial nail model.

They showed that bioglass or hydrogel without antibiotics produced no reduction in infection, but vancomycin-loaded hydrogel could eradicate established infection.


It has been widely claimed that bioglass has the advantage of reducing the risk of antimicrobial resistance.

This paper shows that cases treated with bioglass required significant additional systemic antimicrobial therapy after repeated surgery or recurrence.

It has been shown that repeated surgery promotes change in bacterial cultures (Rupp et al., 2020) [6].

Recurrent, and often suboptimal, systemic therapy drives selection pressure and multi-drug resistance.

There is no evidence that local antimicrobial therapy contributes to this.

The recent study of Bidossi et al. ( 2020) [7] demonstrated that prolonged exposure to high-dose local antibiotics in a ceramic carrier was not associated with any adaptations in bacteria producing antimicrobial resistance.

In contrast to bioglass, the use of local antimicrobials may allow reduced systemic therapy and better antibiotic stewardship (Masrouha et al., 2018; Dudareva et al., 2019) [8][9].


Steinhausen et al. ( 2021) [1] also claim that “The tolerability of BAG is even described as superior when compared to other bone substitutes”.

We cannot find any evidence for this statement in their paper.

When comparing to the paper they quote as evidence (McNally et al., 2016) [10], the claim is not supported. If we compare recurrence rate, reoperation rate, failure of bone healing and amputation rate between the two papers, they report 29%, 47%, 23% and 6% respectively.

In our study of 100 cases, the figures were 4%, 3%, 13% and 0%.

We believe these outcomes would be more tolerable for patients.


Perhaps the most important conclusion from this paper is that serial debridement, followed by defect filling without local antimicrobials, is not a very effective treatment.

We would not recommend use of a hip or knee prosthesis with a 29% failure rate within 2 years.

This problem was identified in the 1970s and prompted the use of antibiotic-loaded PMMA beads by Klemm and Buchholz (Klemm, 1993) [11].

There is now encouraging evidence around the use of single-stage surgery, facilitated by modern local antibiotic carriers, with few complications and high infection eradication rates, even in the most complex cases (Masrouha et al., 2018; McNally et al., 2016; Ferguson et al., 2014; Lam et al., 2019; Pincher et al., 2019; Lorentzen et al., 2020; Drampalos et al., 2018; Jiang et al., 2020; Pesch et al., 2020; Hutting et al., 2021; Mifsud et al., 2020; Zhou et al., 2021) [8][10][12][13][14][15][16][17][18][19][20][21].


We read Martin McNally’s Letter-to-the-Editor [1] with great interest and are pleased to make precise the presentation of our results.

First, we will describe the differences between the patient collectives of the Zeitschrift für Orthopädie und Unfallchirurgie (ZOU) paper [2] and the current Journal of Bone and Joint Infection (JBJI) paper [3].

The inclusion criteria, the enrolment period and the follow-up period of the studies were different.

Fifty patients were treated in the ZOU paper.

Of these, 46 patients were treated with bioactive glass (BAG) and 4 patients with BAG in combination with autologous bone graft.

Ninety-five patients were treated in the JBJI paper.

Of these, 51 patients were treated with BAG, 12 patients with BAG in combination with autologous bone graft and 32 patients with autologous bone graft only.

Thirty of 50 patients (ZOU paper) were excluded for the JBJI paper because they did not meet the additional inclusion criterion “follow-up of at least 12 months” in the JBJI paper.

Therefore, 20 single individuals are published in both the JBJI and ZOU papers.

In these 20 patients, the follow-up data of 14 patients differ in the two publications.

The patients in the JBJI paper have a longer follow-up due to the 18-month longer follow-up period (until January 2018 instead of June 2016).

Six patients did not show up for further follow-up.

The first study (ZOU) is to be regarded as a study with preliminary results.

All patients in whom we implanted bioactive glass up to February 2016 were analyzed, even if they only had a follow-up of 2 months.

This is also listed as limitations in the ZOU paper.

We assumed the two studies to be independent of each other, with a new data set in the JBJI paper: preliminary results in the ZOU paper, longer follow-up in the JBJI paper, no control group in the ZOU paper.

Therefore, we presented the results of the ZOU study in Table 4 in the JBJI paper.

However, we agree that the ZOU paper should have been referenced in the methods section and in Table 4, given the connection between the two publications.


In order to be able to compare complication rates, the patient collectives must be comparable on the one hand, and the definitions must match on the other hand.

A well-known problem of the diagnosis “osteomyelitis” is the extreme heterogeneity.

Reinfection rates of 0 % up to 32.8 % are reported in the literature, depending on the definition and the treatment algorithm [4][5].

In the JBJI study, there is a higher number of patients with complex and therapy-refractory courses compared to the ZOU paper.

In particular, patients of the JBJI study had a longer – unsuccessful – course of disease with an average of 33.8 months from accident to defect filling with bioactive glass, while patients of the ZOU study had an average of 21.5 months.

There may be a negative bias due to our retrospective study design: patients without complications or reinfection may not have visited during our consultation hours any more.

Thus, patients without reinfection have more frequently a follow-up of < 12 months and therefore could not be included in the current JBJI study.

In addition, the definition of reinfection or persistence of infection used by other authors is less strict than ours.

Other authors [6][7] define a “fair” outcome as a wound with prolonged drainage or serum leakage of up to 6 weeks.

These findings would be interpreted as persistent infection/reinfection in our assessment.

The differences in definition as well as in the patient collective may explain the increased complication and reinfection rate in the JBJI study.

Overall, the comparability of our two studies is limited, just like the comparability of complication rates with other studies.


As Martin McNally and we state, there are no large randomized trials directly comparing the use of bioactive glass.

In our opinion, results of animal or in vitro studies – even if they are of high quality – are not sufficient to compare with the results of in vivo studies.

However, there are studies that prove the antibacterial effect of BAG without addition of local antibiotics.

Van Vugt et al. [ 8] evaluated the use of various bone graft substitutes in a systematic review.

No significant differences were found between the different bone graft substitutes (with and without adjunctive use of local antibiotics).

According to our standard at that time, we used the synergy of the local antimicrobial effect of bioactive glass and systemic antibiotics according to the antibiotic resistance pattern to achieve optimal efficacy.

A change in pathogens was seen in only 2 % of our BAG patients.

Independent of the effectiveness, some authors describe the tolerability of BAG as excellent.

To our knowledge, there have been no reports of bioactive glass-associated complications.

This corresponds to our experiences.

Therefore, we consider the tolerability of BAG to be excellent.

This statement in our discussion section does not refer to the patient’s outcome (recurrence rate, failure of bone healing, amputation rate).

In contrast to the excellent tolerability of BAG, our – unpublished – individual experiences with an absorbable gentamicin-loaded calcium sulfate/hydroxyapatite biocomposite were different.

Its use resulted in long-lasting drainage of white fluid like the liquefied bone substitute in most of our patients.

This phenomenon of increased drainage of white fluid is also described by other authors using this bone substitute [9][10].


Lastly, Martin McNally concludes that serial debridement followed by defect filling without local antibiotics is not an effective treatment.

We discussed the one-stage versus two-stage procedure in our JBJI paper.

In fact, several authors highlight the possibility of the one-stage use of BAG as well as of other bone graft substitutes.

However, our standard was a two-stage procedure during the inclusion period of the JBJI study (July 2013 to January 2017).

In addition, our influence of the increased number of previous operations is limited.

Most of our patients had already been treated in other hospitals before, and many of the previous operations were required due to large soft tissue defects.

Data availability.

The data sets analyzed during the current study are available from the corresponding author on reasonable request.


The Gene Expression Omnibus (GEO) repository was established in 2000 (1) to host and freely disseminate high-throughput gene expression data.

The database is built and maintained by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine (NLM), located on the campus of the National Institutes of Health (NIH) in Bethesda, MD, USA.

The data are contributed by the research community, often in compliance with grant or journal directives that require data to be made publicly available, thus allowing others to access, evaluate and re-analyze results.


The three principal objectives of this project are to:


(i) provide a robust, versatile database in which to effi- ciently store high-throughput functional genomic data; (ii) offer the simplest submission procedures and for- mats that support complete and well-annotated data deposits from the research community; and (iii) provide user-friendly mechanisms that allow users to query, locate, review and download experiments of interest.


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In recent years, microarray technology has seen an explo- sion of applications that go far beyond analyzing gene expression levels.

Examples of such studies include those that examine genome single nucleotide polymorphism (SNP) and copy number variations (commonly called 'array comparative genomic hybridization', or aCGH), genome-protein binding surveys (commonly called 'chromatin immunoprecipitation on chip', or ChIP-chip), and various epigenomic factors such as nucleosomal posi- tioning and genome methylation status.

Additionally, non-array-based methodologies such as next-generation sequencing are increasingly being applied to such genome-wide investigations (2).


Despite the fact that GEO was initially set up to store gene expression data generated by microarrays and serial analysis of gene expression (SAGE), the flexible design of the database allows these non-expression or alternative high-through- put data types to be similarly hosted with little extra effort or overhead.

Thus, we have been accommodating to requests to accept such data and extended the minimal standards to fit these types.

In fact, at the time of writing, over 15% of the data in GEO are non-expression data.

Consequently, the name of the database, Gene Expression Omnibus, has become somewhat misleading and perhaps confusing to users.


All experi- ments in GEO and Omix have been newly assigned into broad experiment types, making it much easier for users to locate specific data or technology types.


GEO recently began processing high-throughput sequence data submissions (5-7).

It can be expected that next- generation sequencing technologies will become widely used and have a considerable impact on genome-wide sur- veys (2).

GEO accepts sequence data for studies that examine gene expression, gene regulation, epigenetics, or other studies where measuring sequence abundance is cen- tral to the experiment design.

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As discussed in the Introduction section, the GEO data- base archives a wide variety of rapidly evolving, large- scale, functional genomic experiment types.

These experi- ments generate data of many different file types, formats and content which consequently present considerable challenges in terms of data handling and querying.

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