Free-style propeller ulnar artery perforator flaps for radial forearm flap donor site repair.

INTRODUCTION: The radial forearm free flap (RFFF) is still one of the most used free flaps to repair soft tissue defects of the head and neck. Among its main drawbacks, it presents severe donor site complications. We report our experience about the use of free-style propeller ulnar artery perforator flaps (UAP) to repair RFFF donor site. PATIENTS AND METHODS: From February 2010 to June 2020, six patients who underwent immediate tongue reconstruction with RFFF after cancer excision, had the donor site at the forearm reconstructed with a free-style propeller UAP flap. The indication for a UAP flap was based on defect size and the presence of tendons or radial nerve exposure. Ulnar artery perforators were identified intra-operatively with a handheld Doppler. The UAP flaps were harvested and rotated to cover donor site defects. The patients' mean age was 59, ranging from 49 to 65 years old. The defects size ranged from 8-12 cm × 5-7 cm with a mean size of 10.5 × 6.7 cm. RESULTS: The UAP flap harvested ranged from 8-11 cm × 5-7 cm with a mean size of 10.5 × 5.5 cm. The perforators, identified with a power Doppler, were located at the middle third of the forearm. Flaps' rotation varied from 90 to 160° with a mean rotation of 122°. Mean operating time was 60 min ranging from 40 to 75 min for UAP flap elevation. No flap necrosis or tendon exposure occurred. One case of wound dehiscence was reported. Two out of six patients suffered from tendon adhesions to the flap. UAP flap donor site was primarily closed in four out of six patients, whereas two cases required a split-thickness skin grafts. Mean donor site healing time was about 20 days (19.8 days) ranging from 14 to 30 days. Follow-up ranged from 12 to 31 months with a mean follow up time of 19 months (18.6 months). At 6 months follow-up only one patient experienced a functional limitation of wrist and finger joints extension of 20° which required tenolysis. At the end of the patient's follow up that is 22 months, the range of movement was within normal limits. In our casuistry neuropathic pain was absent. CONCLUSIONS: RFF is still a paramount tool in reconstructive surgery but its donor site is still burdened by a high complication rate. Free-style UAP flaps can provide a local and safe solution.

Immediate Soft Tissue Reconstruction in Lower Limb Traumas Using Propeller Perforator Flaps.

BACKGROUND: Open bone fractures represent a demanding clinical condition that can be life- or limb-threatening. For small- to medium-size soft tissue defects of the lower extremity, propeller perforator flaps are a viable option for reconstruction. We report our experience in Gustilo IIIB open fractures treatment with immediate coverage through propeller-based perforator flaps performed in acute setting. METHODS: Between 2019 and 2022, 94 Gustilo III fractures were admitted to our trauma center, and 28 were Gustilo IIIB fractures. Five patients were eligible for an immediate perforator-based propeller flaps reconstruction. The mean age of the patients was 33.8 years. All of them were male. One of them was a heavy smoker (15 cigarettes per day). No other comorbidities were present. RESULTS: The mean operating time, including fracture reduction and fixation time, was 106 minutes. The length of the defect varied between 5 and 7 cm and the width between 3 and 4 cm. We reported no major complications, and none of the patients required a surgical revision of the flap. A patient with smoking habit reported a superficial flap necrosis. The mean follow-up was 5.5 months. No cases of nonunion or osteomyelitis have developed so far. CONCLUSIONS: The use of perforator-based propeller flaps, in acute trauma setting, has not been reported in literature so far. In our experience, propeller perforator flaps are a viable reconstructive option in immediate reconstruction during the acute management of open fractures.

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern.

The emergence of a polybasic cleavage motif for the protease furin in SARS-CoV-2 spike has been established as a major factor for human viral transmission. The region N-terminal to that motif is extensively mutated in variants of concern (VOCs). Besides furin, spikes from these variants appear to rely on other proteases for maturation, including TMPRSS2. Glycans near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, we identify that sialic acid-containing O-linked glycans on Thr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage and posit O-linked glycosylation as a likely driving force for the emergence of VOC mutations. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, an event that is suppressed by mutations in the VOCs Alpha, Delta, and Omicron. We found that the sole incorporation of N-acetylgalactosamine did not impact furin activity in synthetic O-glycopeptides, but the presence of sialic acid reduced the furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on TMPRSS2 cleavage. With a chemistry-centered approach, we substantiate O-glycosylation as a major determinant of spike maturation and propose disruption of O-glycosylation as a substantial driving force for VOC evolution.

Posterior Circumflex Humeral Artery Perforator Flap: A Cadaveric Study and Case Series.

BACKGROUND: Reconstruction of soft tissue defects of the acromioclavicular region represents a relatively uncommon but challenging event. Many muscular, fasciocutaneous, and perforator flaps have been described, including the posterior circumflex humeral artery perforator (PCHAP) flap based on the direct cutaneous perforator of the PCHA. This study aims to describe a variant of the PCHAP flap, based on a constant musculocutaneous perforator, by means of a cadaveric study and a case series. METHODS: A cadaveric study was conducted using 11 upper limbs. The perforator vessels originating from the PCHA were dissected and the musculocutaneous ones were identified and measured in their length and distance from the deltoid tuberosity. Besides, we retrospectively analyzed the posterior shoulder reconstruction conducted among 2 plastic surgery department (San Gerardo Hospital, Monza and Hospital Papa Giovanni XXIII, Bergamo) using the musculocutaneous perforators of the PCHA. RESULTS: The cadaver dissection showed the presence of a constant musculocutaneous perforator arising from the PCHA. The mean pedicle length is 6.10 ± 1.18 cm, and the musculocutaneous perforator pierces the fascia at a mean distance of 10.4 ± 2.06 cm from the deltoid tuberosity. In all the cadaver dissected, the perforator of interest divided into 2 terminal branches, anterior and posterior, nourishing the skin paddle.In our case series, the mean age of the patients was 66.7 years, the mean size of the defect was 46 cm 2 , the mean operating time was 79.3 minutes, the mean length of hospital stay was 2.7 days, and the complication rate was 28.6%. CONCLUSIONS: According to this preliminary data, the PCHAP flap based on the musculocutaneous perforator seems to be a reliable alternative in posterior shoulder region reconstruction.

Cell-specific bioorthogonal tagging of glycoproteins.

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

Thoracodorsal artery perforator flap, muscle-sparing latissimus dorsi, and descending branch latissimus dorsi: A multicenter retrospective study on early complications and meta-analysis of the literature.

The thoracodorsal (TD) vessels represent a complex vascular system that offers a variety of pedicled and free flaps. Variations of the classical latissimus dorsi (LD) flap have been developed to overcome its major drawbacks. The thoracodorsal artery perforator (TDAP) flap described by Angrigiani represents one of these options. Other techniques have been defined as "muscle-sparing" latissimus dorsi (MSLD) due to the preservation of the LD muscle and the TD nerve, in whole or in part. Nevertheless, the term "muscle sparing" has also been applied to the descending branch LD (DB-LD) flap which requires the denervation of the LD muscle. According to our knowledge, there are no articles in the literature reviewing and comparing the reconstructive options based on the TD vessels. We performed a systematic search in PubMed, Web of Science, and Cochrane databases to perform a literature review and meta-analysis about the reconstructive options based on the TD vessels. The primary outcome of interest was the percentage of flaps developing a specific early complication, i.e., hematoma of the donor site, seroma of the donor site, partial flap loss, total flap loss, wound dehiscence, and wound infection. Moreover, we analyzed the outcomes and complications of our cases, comparing the MSLD flaps, the DB-LD flaps, and the TDAP flaps. According to both our casuistry and the literature, the three techniques can be considered safe in terms of early donor site complications. According to the literature, MSLD has been shown to develop partial flap necrosis more frequently than the TDAP flap.

A heterodox defense of the actualist higher-order thought theory.

I defend the actualist higher-order thought theory against four objections. The first objection contends that the theory is circular. The second one contends that the theory is unable to account for the alleged epistemic position we are in with respect to our own conscious mental states. The third one contends that the theory is unable to account for the evidence we have for the proposition that all conscious mental states are represented. The fourth one contends that the theory does not accommodate the intimacy we have with our own conscious mental states. To some extent, my defense will be heterodox, in the sense that I will show that some objections are satisfactorily answerable even if we concede to the objectors a point that higher-order theorists do not seem to be willing to concede, that is, that the theory is the result of conceptual analysis.

Enzymatic elaboration of oxime-linked glycoconjugates in solution and on liposomes.

Oxime formation is a convenient one-step method for ligating reducing sugars to surfaces, producing a mixture of closed ring α- and ß-anomers along with open-chain (E)- and (Z)-isomers. Here we show that despite existing as a mixture of isomers, N-acetylglucosamine (GlcNAc) oximes can still be substrates for ß(1,4)-galactosyltransferase (ß4GalT1). ß4GalT1 catalysed the galactosylation of GlcNAc oximes by a galactose donor (UDP-Gal) both in solution and in situ on the surface of liposomes, with conversions up to 60% in solution and ca. 15-20% at the liposome surface. It is proposed that the ß-anomer is consumed preferentially but long reaction times allow this isomer to be replenished by equilibration from the remaining isomers. Adding further enzymes gave more complex oligosaccharides, with a combination of α-1,3-fucosyltransferase, ß4GalT1 and the corresponding sugar donors providing Lewis X coated liposomes. However, sialylation using T. cruzi trans-sialidase and sialyllactose provided only very small amounts of sialyl Lewis X (sLex) capped lipid. These observations show that combining oxime formation with enzymatic elaboration will be a useful method for the high-throughput surface modification of drug delivery vehicles, such as liposomes, with cell-targeting oligosaccharides.

A radical relationist solution to the problem of intentional inexistence.

The problem of intentional inexistence arises because the following (alleged) intuitions are mutually conflicting: it seems that sometimes we think about things that do not exist; it seems that intentionality is a relation between a thinker and what such a thinker thinks about; it seems that relations entail the existence of what they relate. In this paper, I argue for what I call a radical relationist solution. First, I contend that the extant arguments for the view that relations entail the existence of their relata are wanting. In this regard, I defend a kind of pluralism about relations according to which more than one kind of relation involves non-existents. Second, I contend that there are reasons to maintain that all thoughts are relations between thinkers and the things they are about. More accurately, I contend that the radical relationist solution is to be preferred to both the intentional content solution (as developed by Crane) and the adverbial property solution (as developed by Kriegel). Finally, I argue that once the distinction between thinking "X" and thinking about X has been drawn, the radical relationist solution can handle issues like ontological commitment, substitutivity failure, scrutability, and non-specificity.

Intraoperative near-infrared spectroscopy for pedicled perforator flaps: a possible tool for the early detection of vascular issues.

BACKGROUND: Pedicled perforator flaps can present postoperative complications similar to those encountered in free flap surgery. Beyond a clinical evaluation, there is still no reliable technical aid for the early prediction of vascular issues. The aim of this study was to assess the support of near-infrared spectroscopy technology as an intraoperative tool to anticipate postsurgical flap ischemia. METHODS: We prospectively enrolled 13 consecutive patients who were referred to our hospital from March 2017 to July 2018 and required a reconstructive procedure with a pedicled fasciocutaneous perforator flap. We measured flap peripheral capillary oxygen saturation (SpO2) in each patient with a Somanetics INVOS 5100C Cerebral/Somatic Oximeter (Medtronic), both before and after transposition. Patient demographics, operative data, and complications were then recorded during the following 6 months. We analyzed the data using the Wilcoxon signed-rank test and linear regression. RESULTS: The mean flap SpO2 before and after transposition was 92%±3% and 78%±19%, respectively. The mean change in SpO2 was 14%±17%, with a range of 0% to 55%. The change in saturation and mean saturation ratio were significantly different between patients with and without postoperative flap necrosis. CONCLUSIONS: An immediate quantitative analysis of flap peripheral capillary SpO2 after transposition has never before been described. In our experience, an intraoperative drop in SpO2 equal to or greater than 15%-20% predicted vascular complications in pedicled perforator flaps. Conversely, flap size and rotation angle were not correlated with the risk of flap necrosis.

A promiscuous glycosyltransferase generates poly-ß-1,4-glucan derivatives that facilitate mass spectrometry-based detection of cellulolytic enzymes.

Promiscuous activity of a glycosyltransferase was exploited to polymerise glucose from UDP-glucose via the generation of ß-1,4-glycosidic linkages. The biocatalyst was incorporated into biocatalytic cascades and chemo-enzymatic strategies to synthesise cello-oligosaccharides with tailored functionalities on a scale suitable for employment in mass spectrometry-based assays. The resulting glycan structures enabled reporting of the activity and selectivity of celluloltic enzymes.

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses.

Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogues that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analogue glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal reporter reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

Wide Local Excisions and Pedicled Perforator Flaps in Hidradenitis Suppurativa: A Study of Quality of Life.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic skin pathology characterized by the occurrence of inflamed and swollen lesions, with a devastating impact of the disease on patient's quality of life. Wide local excision (WLE) of the affected tissue is the criterion-standard treatment, but there is no general consensus on the best reconstructive technique. The aim of this article is to evaluate the quality of life before and after WLE combined with pedicled perforator flap reconstruction, in patients suffering from HS. METHODS: We analyzed 26 consecutive patients suffering from Hurley stage III HS. Every patient underwent WLE, followed by reconstruction with pedicled perforator flap. Dermatology Quality of Life Index (DLQI) tests were administered to every patient preoperatively and 6 months after surgery, when the clinical condition was considered stable. Dermatology Quality of Life Index scores were compared in terms of means with Student t test. Linear regression studies were used to compare the changes in DLQI score with the surgical and clinical variables. RESULTS: Axillary area involvement was found in 21 of 27 patients; 6 patients had more than 1 body region involved. Thirty-two pedicled perforator flaps were performed, and 22 were thoracodorsal artery perforator flaps. Mean DLQI test score before the first operation was 21.31 ± 4.79; the average DLQI score after the last follow-up was 5 ± 2.95 (P < 0.0001). Dermatology Quality of Life Index scores were not influenced by complications including reoperation. There were also no correlations found between DLQI score delta and number of reoperation (-0.18) or days of hospital stay (-0.13). CONCLUSIONS: Wide local excision followed by pedicled perforator flap reconstruction allows a radical excision of HS areas with short postoperative healing periods. Dermatology Quality of Life Index scores confirmed high levels of patients' satisfaction. Disadvantages of this technique include difficult learning curve, long operating time, and a nonnegligible complications rate.

Enzymatic Building-Block Synthesis for Solid-Phase Automated Glycan Assembly.

Automated chemical oligosaccharide synthesis is an attractive concept that has been successfully applied to a large number of target structures, but requires excess quantities of suitably protected and activated building blocks. Herein we demonstrate the use of biocatalysis to supply such reagents for automated synthesis. By using the promiscuous NmLgtB-B ß1-4 galactosyltransferase from Neisseria meningitidis we demonstrate fast and robust access to the LacNAc motif, common to many cell-surface glycans, starting from either lactose or sucrose as glycosyl donors. The enzymatic product was shown to be successfully incorporated as a complete unit into a tetrasaccharide target by automated assembly.

Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides.

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.

Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

Automated glycan assembly of Streptococcus pneumoniae type 14 capsular polysaccharide fragments.

S. pneumoniae is a major human pathogen with increasing antibiotic resistance. Pneumococcal vaccines consist of capsular polysaccharide (CPS) or their related fragments conjugated to a carrier protein. The repeating unit of S. pneumoniae type 14 CPS shares a core structure with the CPS of Group B Streptococcus (GBS) type III: the only difference is that the latter exhibits a sialic acid unit, with a α-2,3 linkage to galactose. Here, the automated glycan assembly (AGA) of two frameshifts of the repeating unit of S. pneumoniae type 14 is described. The same strategy is used to assemble dimers of the different repeating unit frameshifts. The four structures are assembled with only three commercially available monosaccharide building blocks. We also report an example of how enzymatic sialylation of the compounds obtained with AGA completes a synthetic route for GBS type III glycans. The synthesized structures were tested in competitive ELISA and further confirmed the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc to be the minimal epitope of S. pneumoniae type 14.

Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from Photobacterium damselae To Generate a Highly Sialylated Recombinant α-1-Antitrypsin.

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from Photobacterium damselae can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the P. damselae in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the in vitro activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.