Pedicled palmar intermetacarpal perforator flap for skin-graftless syndactyly release: Anatomical study and clinical application.

INTRODUCTION: Dorsal hand skin flaps have been described in order to avoid digit skin grafting in syndactyly release. Although these skin-graftless techniques present a lower rate of long term complications, they result in unsightly dorsal scarring and worse patient satisfaction. We describe a novel technique using an intermetacarpal palmar flap by performing an anatomic study and clinical application. MATERIALS AND METHODS: Ten colored-latex-injected fresh upper limbs were used to study the palmar cutaneous perforators to second to fourth intermetacarpal space skin flaps designed elliptical, its width extending from the center of each involved ray and measuring 40%-50% the length of the palm. RESULTS: The anatomical study revealed a mean 2.77 (range 1 to 4) cutaneous perforators originating from either the common or proper digital vessels for every intermetacarpal space and measuring 0.4 mm (range 0.3 to 0.5 mm) in diameter. CLINICAL APPLICATION: This flap, measuring a mean of 21.5 mm (range 20 to 23 mm) in length and 9.5 mm (range 9 to 10 mm) in width, was used to release simple syndactylies in three patients of mean age 24 months (range = 18-30 months). After skin incision, cutaneous septa to the digital canal were released to permit flap advancement to the web space. Cutaneous perforators were not dissected. Intra-operative palmar-flap advancement provided commissure coverage in all children with no skin grafts needed. Over a mean follow-up of 15 months, no complications occurred, mean palm scar VSS was 2.4 (range 2 to 3) while it was 3 (range 3 to 3) for the commissure scars. CONCLUSIONS: Skin-graftless syndactyly release using a palmar intermetacarpal flap seems both reliable and easy-to-perform. LEVEL OF EVIDENCE: Therapeutic IV.

COVID-19 diagnosis by RT-qPCR in alternative specimens.

BACKGROUND: The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES: The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS: We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS: When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS: Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.

Remote Activation of Hollow Nanoreactors for Heterogeneous Photocatalysis in Biorelevant Media.

Major current challenges in nano-biotechnology and nano-biomedicine include the implementation of predesigned chemical reactions in biological environments. In this context, heterogeneous catalysis is emerging as a promising approach to extend the richness of organic chemistry onto the complex environments inherent to living systems. Herein we report the design and synthesis of hybrid heterogeneous catalysts capable of being remotely activated by near-infrared (NIR) light for the performance of selective photocatalytic chemical transformations in biological media. This strategy is based on the synergistic integration of Au and TiO2 nanoparticles within mesoporous hollow silica capsules, thus permitting an efficient hot-electron injection from the metal to the semiconductor within the interior of the capsule that leads to a confined production of reactive oxygen species. These hybrid materials can also work as smart NIR-responsive nanoreactors inside living mammalian cells, a cutting-edge advance toward the development of photoresponsive theranostic platforms.

Bioorthogonal Azide-Thioalkyne Cycloaddition Catalyzed by Photoactivatable Ruthenium(II) Complexes.

Tailored ruthenium sandwich complexes bearing photoresponsive arene ligands can efficiently promote azide-thioalkyne cycloaddition (RuAtAC) when irradiated with UV light. The reactions can be performed in a bioorthogonal manner in aqueous mixtures containing biological components. The strategy can also be applied for the selective modification of biopolymers, such as DNA or peptides. Importantly, this ruthenium-based technology and the standard copper-catalyzed azide-alkyne cycloaddition (CuAAC) proved to be compatible and mutually orthogonal.

Response to direct-acting antivirals for hepatitis C treatment in vertically HIV/HCV co-infected patients.

Direct-acting antivirals (DAAs) for HCV treatment have improved tolerance and efficacy among adults, but experience in vertical transmission is scarce. In our vertically HIV/HCV co-infected youth cohort of 58 patients, DAA achieved excellent rates of cure among naïve and pretreated individuals. Treating vertically infected seems important as 29.6% displayed advanced fibrosis at treatment initiation.

A Randomized Clinical Trial on the Use of Antiseptic Solutions for the Pin-Site Care of External Fixators: Chlorhexidine-Alcohol Versus Povidone-Iodine.

Pin-site infections remain a common clinical complication in patients with external fixators. Pin-site care is commonly performed with either chlorhexidine-alcohol solution or povidone-iodine solution. This study aimed to investigate the superiority of chlorhexidine-alcohol solution versus povidone-iodine solution for external fixator pin-site care in pin-site infection. This prospective randomized clinical trial using an open, parallel-group design was conducted in a single Spanish hospital. Eligible consenting patients from November 2018 to May 2019 who underwent placement of an external fixator were included. Patients were randomly assigned to receive pin-site care using either a 2% chlorhexidine-alcohol solution or a 10% povidone-iodine solution. The primary endpoint was the development of a pin-site infection. In total, 568 pins were analyzed (128 patients, with a mean of 4.3 pins per patient). No significant differences were found between groups. However, statistically significant differences were found regarding time and infection variables. The longer the person had the fixator, the higher the risk of infection, t(x) = 5.49, p = .002. Both chlorhexidine-alcohol and povidone-iodine solutions are equally effective antiseptic agents for the prevention of infections in external fixators.

Anion Recognition as a Supramolecular Switch of Cell Internalization.

The cell internalization of designed oligoarginine peptides equipped with six glutamic acid residues and an anionic pyranine at the N-terminus is triggered upon addition of a supramolecular host. This host binds specifically to the pyranine moiety, enabling the complex to traverse the cell membrane. Interestingly, none of the components, neither the host nor the guest, are able to cross the cell membrane on their own.

From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors.

Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis.

Ruthenium-Catalyzed Azide-Thioalkyne Cycloadditions in Aqueous Media: A Mild, Orthogonal, and Biocompatible Chemical Ligation.

The development of efficient metal-promoted bioorthogonal ligations remains as a major scientific challenge. Demonstrated herein is that azides undergo efficient and regioselective room-temperature annulations with thioalkynes in aqueous milieu when treated with catalytic amounts of a suitable ruthenium complex. The reaction is compatible with different biomolecules, and can be carried out in complex aqueous mixtures such as phosphate buffered saline, cell lysates, fetal bovine serum, and even living bacteria (E. coli). Importantly, the reaction is mutually compatible with the classical CuAAC.

Sequence-dependent internalization of aggregating peptides.

Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 µm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease.

Homeopathic medicines for prevention of influenza and acute respiratory tract infections in children: blind, randomized, placebo-controlled clinical trial.

BACKGROUND: Influenza and its complications are common at all ages, especially in children. Vaccines and anti-influenza drugs aim to prevent it. Preventative approaches with favorable risk profiles should be considered for flu, particularly since the evidence of the efficacy of anti-viral drugs is debated. METHODS: This pragmatic clinical trial was conducted in the Brazilian Public Health System in Petrópolis (BPHSP) with children aged from 1 to 5 years old. The medications used were mainly selected based on in vitro experiments (InfluBio), and in successful qualitative clinical experiences (Homeopathic Complex). Following informed parental consent, subjects were randomly distributed, in a blind manner, to three experimental groups: Homeopathic Complex, Placebo, and InfluBio. BPHSP health agents collected flu and acute respiratory infection symptomatic episodes monthly following the established protocol. The number of these episodes was registered in one year (2009-2010). RESULTS: Out of the 600 children recruited, 445 (74.17%) completed the study (149: Homeopathic complex; 151: Placebo; 145: InfluBio). The number of flu and acute respiratory infection symptomatic episodes detected in this clinical trial was low; however, it was different between homeopathic groups and placebo (p < 0.001). In the first year post-intervention, 46/151 (30.5%) of children in the placebo group developed 3 or more flu and acute respiratory infection episodes, while there was no episode in the group of 149 children who used Homeopathic Complex, and only 1 episode in the group of 145 (1%) children who received InfluBio. CONCLUSION: These results suggested that the use of homeopathic medicines minimized the number of flu and acute respiratory infection symptomatic episodes in children, signalizing that the homeopathic prophylactic potential should be investigated in further studies.

Homeopathic treatments modify inflammation but not behavioral response to influenza antigen challenge in BALB/c mice.

BACKGROUND: Influenza affects thousands of people worldwide every year, motivating the development of new therapies. In this work, the effects of two homeopathic preparations (influenza biotherapies and thymulin) were chosen following two different rationales: isotherapy and endo-isotherapy models. The homeopathic effects were evaluated individually considering the inflammatory and behavioral responses against influenza virus antigen were studied in BALB/c mice. METHODS: Male adult mice were treated orally and blindly for 21 days with highly diluted influenza virus or with thymulin, and were divided in two sets of experiments. The first series of experiments aimed to describe their behavior, using an open field (OF) device. In the second series, mice were challenged subcutaneously with influenza hemagglutinin antigen (7 µg/200 µl) at day 21. At day 42, behavior and inflammation response were evaluated. RESULTS: No behavioral changes were seen in OF tests at any time point after treatments. Flow cytometry and morphometry revealed significant changes in T and B cell balance after influenza antigen challenge, varying according to treatment. CONCLUSION: The results show that both homeopathic treatments induced subtle changes in acquired immune anti-viral response regulation. A deeper understanding of the mechanism could elucidate their possible use in influenza epidemiological situations.

Ruthenation of Non-stacked Guanines in DNA G-Quadruplex Structures: Enhancement of c-MYC Expression.

Guanine quadruplexes (GQs) are compact four-stranded DNA structures that play a key role in the control of a variety of biological processes, including gene transcription. Bulky ruthenium complexes featuring a bipyridine, a terpyridine, and one exchangeable ligand ([Ru(terpy)(bpy)X]n+ ) are able to metalate exposed guanines present in the GQ of the c-MYC promoter region that are not involved in quadruplex base pairing. qRT-PCR and western-blot experiments indicated that the complexes promote a remarkable increase in the expression of this oncogene. We also show that exchangeable thioether ligands (X=RSR', Met) allow regulation of the metalating activity of the complex with visible light.

Coronin 1A promotes a cytoskeletal-based feedback loop that facilitates Rac1 translocation and activation.

The activation of the Rac1 GTPase during cell signalling entails its translocation from the cytosol to membranes, release from sequestering Rho GDP dissociation inhibitors (RhoGDI), and GDP/GTP exchange. In addition to those steps, we show here that optimal Rac1 activation during cell signalling requires the engagement of a downstream, cytoskeletal-based feedback loop nucleated around the cytoskeletal protein coronin 1A and the Rac1 exchange factor ArhGEF7. These two proteins form a cytosolic complex that, upon Rac1-driven F-actin polymerization, translocates to juxtamembrane areas where it expands the pool of activated, membrane-bound Rac1. Such activity requires the formation of an F-actin/ArhGEF7-dependent physical complex of coronin 1A with Pak1 and RhoGDIα that, once assembled, promotes the Pak1-dependent dissociation of Rac1 from the Rac1/RhoGDIα complex and subsequent Rac1 activation. Genetic evidence demonstrates that this relay circuit is essential for generating sustained Rac1 activation levels during cell signalling.

De Novo Engineering of Pd-Metalloproteins and Their Use as Intracellular Catalysts.

The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactivation. We now demonstrate that grafting Pd(II) complexes into engineered ß-sheets of a model WW domain results in cell-compatible palladominiproteins that effectively catalyze depropargylation reactions inside HeLa cells. The concave shape of the WW domain ß-sheet proved particularly suitable for accommodating the metal center and protecting it from rapid deactivation in the cellular environment. A thorough NMR and computational study confirmed the formation of the metal-stapled peptides and allowed us to propose a three-dimensional structure for this novel metalloprotein motif.

Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy.

BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

α-Galactosidase aggregation is a determinant of pharmacological chaperone efficacy on Fabry disease mutants.

Fabry disease is a lysosomal storage disorder caused by loss of α-galactosidase function. More than 500 Fabry disease mutants have been identified, the majority of which are structurally destabilized. A therapeutic strategy under development for lysosomal storage diseases consists of using pharmacological chaperones to stabilize the structure of the mutant protein, thereby promoting lysosomal delivery over retrograde degradation. The substrate analog 1-deoxygalactonojirimycin (DGJ) has been shown to restore activity of mutant α-galactosidase and is currently in clinical trial for treatment of Fabry disease. However, only ∼65% of tested mutants respond to treatment in cultured patient fibroblasts, and the structural underpinnings of DGJ response remain poorly explained. Using computational modeling and cell culture experiments, we show that the DGJ response is negatively affected by protein aggregation of α-galactosidase mutants, revealing a qualitative difference between misfolding-associated and aggregation-associated loss of function. A scoring function combining predicted thermodynamic stability and intrinsic aggregation propensity of mutants captures well their aggregation behavior under overexpression in HeLa cells. Interestingly, the same classifier performs well on DGJ response data of patient-derived cultured lymphoblasts, showing that protein aggregation is an important determinant of chemical chaperone efficiency under endogenous expression levels as well. Our observations reinforce the idea that treatment of aggregation-associated loss of function observed for the more severe α-galactosidase mutants could be enhanced by combining pharmacological chaperone treatment with the suppression of mutant aggregation, e.g. via proteostatic regulator compounds that increase cellular chaperone expression.

Impact of agr dysfunction on virulence profiles and infections associated with a novel methicillin-resistant Staphylococcus aureus (MRSA) variant of the lineage ST1-SCCmec IV.

BACKGROUND: A novel variant of the ST1-SCCmecIV methicillin-resistant Staphylococcus aureus (MRSA) lineage, mostly associated with nosocomial bloodstream infections (BSI), has emerged in Rio de Janeiro. Bacterial biofilm has been considered a major virulence factor in central venous catheter-associated BSI. The mechanisms involved in biofilm formation/accumulation are multifactorial and complex. Studies have suggested that biofilm production was affected in vitro and vivo for agr-null mutants of S. aureus. RESULTS: The impact of naturally occurring inhibition of agr signaling on virulence profiles and infections associated with the ST1 variant was investigated. agr dysfunction was detected in a significant percentage (13%) of the isolates with concomitant increase in biofilm accumulation in vitro and in vivo, and enhanced ability to adhere to and invade airway cells. The biofilm formed by these ST1 isolates was ica-independent and proteinaceous in nature. In fact, the improved colonization properties were paralleled by an increased expression of the biofilm-associated genes fnbA, spa and sasG. The transcription of sarA, a positive regulator of agr, was two-times reduced for the agr-dysfunctional MRSA. Remarkably, the agr inhibition was genetically stable. Indeed, agr-dysfunctional isolates succeed to colonize and cause both acute and chronic infections in hospitalized patients, and also to effectively accumulate biofilm in a mouse subcutaneous catheter implant model. CONCLUSION: The ability of agr-dysfunctional isolates to cause infections in humans and to form biofilm in the animal model suggests that therapeutic approaches based on agr-inactivation strategies are unlikely to be effective in controlling human-device infections caused by ST1 isolates. The increased biofilm accumulation associated with the acquisition of multiple antimicrobial resistant traits might have influenced (at least in part) the expansion of this USA400 related clone in our hospitals.

Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.

Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.

Detection of avian paramyxoviruses in migratory and resident birds in the state of Rio de Janeiro, Brazil.

Paramyxoviruses and avian influenza viruses are present worldwide, and wild birds are known natural reservoirs of these viruses. This study monitored the circulation of these viruses in migratory and resident coastal birds captured in the state of Rio de Janeiro, Brazil. In total, 494 birds were trapped, and their fecal samples were collected and inoculated into embryonated chicken eggs. The allantoic fluids were evaluated using a hemagglutination test and PCR amplification of the genes of the M and L proteins of influenza A virus and paramyxovirus, respectively. Avian paramyxovirus was detected in 5 (1.01%) of the birds. The majority of these viruses were isolated from migratory birds classified into the order Charadriiformes (families Scolopacidae and Charadriidae). Four samples were characterized as avian paramyxovirus serotype-2 (APMV-2) by a hemagglutination inhibition test. These results reinforce the importance of continuous surveillance of wild species in Brazil.