Revisiting Bipedicled Flaps for Lower Extremity Distal Third Defects.

BACKGROUND: Lower extremity reconstruction of the distal third of the leg is challenging. Free tissue transfer is the criterion standard. The COVID-19 pandemic encouraged seeking alternatives for resource consuming procedures. Bipedicled flaps are flaps with a dual-source subdermal perfusion. The purpose of this study was to assess outcomes of patients who had bipedicled flaps primary or auxiliary local flap for distal third leg/foot reconstruction. METHODS: A retrospective review of patients undergoing lower extremity reconstruction (2020-2021) was performed. Inclusion criteria were patients older than 18 years with lower extremity wounds secondary to traumatic injury for which bipedicled flaps were used in the reconstruction. Exclusion criteria included lower extremity wounds secondary to peripheral vascular disease or diabetes. RESULTS: Fourteen patients were included in the study. All patients had distal third of the leg/foot wounds, and 12 patients (87.5%) had concurrent leg fractures. In 8 patients (57.1%), the bipedicled flap was used to decrease the wound size and facilitate another concurrent flap: hemisoleus (21.4%), anterior tibialis muscle turnover (14.3%), medial plantar artery (14.3%), and posterior tibial artery perforator (14.3%). Mean wound size for bipedicle flaps used alone was 42.0 ± 18.3 cm2, whereas wounds that required a bipedicled flap with an additional flap were 69.9 ± 80.8 cm2 (P = 0.187). Two patients had partial flap necrosis (14.3%) but healed their defect. One patient had nonunion (7.1%). Limb salvage rate was 100%. CONCLUSIONS: Bipedicled flaps can be used as an alternative to free flaps in distal third leg/foot defects in select patients. If distal extremity wounds cannot be covered with a bipedicled flap alone, the flap can be used an accessory flap to facilitate reconstruction with other local flaps.

Current-Season Infection With Ordinary and Recombinant Strains of Potato virus Y Has Detrimental Effects on Chipping Potato Yield and Tuber Quality.

Potato virus Y (PVY) is the most economically important virus infecting potatoes worldwide. Current-season spread of PVY occurs when aphids transmit the virus from infected to noninfected plants during the growing season. The impact of current-season PVY infection on yield and quality of chip processing potatoes is not well documented. In a replicated, greenhouse experiment conducted over 2 years, we measured the effect of current-season infection with four PVY strains (PVYO, PVYN-Wi, PVYNTN, and PVYN:O) on chip processing varieties Atlantic, Lamoka, and Snowden. PVY infection decreased yield and tuber specific gravity for some combinations of potato variety and virus strain but did not affect the appearance of chips including the prevalence of stem-end chip defects. This work suggests that current-season infection of chipping potatoes imposes a cost on producers and emphasizes the need for continued investment in seed certification and development of PVY-resistant cultivars.

A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation.

The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

Self-organizing hair peg-like structures from dissociated skin progenitor cells: New insights for human hair follicle organoid engineering and Turing patterning in an asymmetric morphogenetic field.

Human skin progenitor cells will form new hair follicles, although at a low efficiency, when injected into nude mouse skin. To better study and improve upon this regenerative process, we developed an in vitro system to analyse the morphogenetic cell behaviour in detail and modulate physical-chemical parameters to more effectively generate hair primordia. In this three-dimensional culture, dissociated human neonatal foreskin keratinocytes self-assembled into a planar epidermal layer while fetal scalp dermal cells coalesced into stripes, then large clusters, and finally small clusters resembling dermal condensations. At sites of dermal clustering, subjacent epidermal cells protruded to form hair peg-like structures, molecularly resembling hair pegs within the sequence of follicular development. The hair peg-like structures emerged in a coordinated, formative wave, moving from periphery to centre, suggesting that the droplet culture constitutes a microcosm with an asymmetric morphogenetic field. In vivo, hair follicle populations also form in a progressive wave, implying the summation of local periodic patterning events with an asymmetric global influence. To further understand this global patterning process, we developed a mathematical simulation using Turing activator-inhibitor principles in an asymmetric morphogenetic field. Together, our culture system provides a suitable platform to (a) analyse the self-assembly behaviour of hair progenitor cells into periodically arranged hair primordia and (b) identify parameters that impact the formation of hair primordia in an asymmetric morphogenetic field. This understanding will enhance our future ability to successfully engineer human hair follicle organoids.

Active nerve management for above the knee amputation: A comparison of through the wound versus posterior approach.

Targeted muscle reinnervation (TMR) and regenerative peripheral nerve interface (RPNI) are used to prevent or treat neuromas in amputees. TMR for above-the-knee amputation (AKA) is most commonly performed through a posterior incision rather than the stump wound because recipient motor nerves are primarily located in the proximal third of the thigh. When preventative TMR is performed with concurrent AKA, a posterior approach requires intraoperative repositioning and an additional incision. The purpose of this study was to evaluate feasibility of TMR and operative times for nerve management performed through the wound compared to a posterior approach in AKA patients to guide surgical decision-making. Patients who underwent AKA with TMR between 2018-2023 were reviewed. Patients were divided into two groups: TMR performed through the wound (Group I) and TMR performed through a posterior approach (Group II). If a nerve was unable to undergo coaptation for TMR due to the lack of suitable donor motor nerves, RPNI was performed. Eighteen patients underwent AKA with nerve management were included from Group I (8 patients) and Group II (10 patients). TMR coaptations performed on distinct nerves was 1.5 ± 0.5 in Group I compared to 2.6 ± 0.5 in Group II (p = 0.001). Operative time for Group I was 200.7 ± 33.4 min compared to 326.5 ± 37.1 min in Group II (p = 0.001). TMR performed through the wound following AKA requires less operative time than a posterior approach. However, since recipient motor nerves are not consistently found near the stump, RPNI may be required with TMR whereas the posterior approach allows for more TMR coaptations.

Turnover Tendon Lengthening Does Not Alter Biomechanical Strength of Pronator Teres-to-Extensor Carpi Radialis Brevis Tendon Transfer.

BACKGROUND: The pronator teres (PT) to extensor carpi radialis brevis (ECRB) tendon transfer reestablishes wrist extension. Occasionally, the PT periosteal extension is of suboptimal quality to support a strong transfer. In these instances, turnover lengthening techniques can increase usable tendon length. This study characterized the optimal length of tendon turnover and the effect of lengthening on transfer strength. METHODS: Twenty-seven cadaveric extensor tendons were lengthened using the turnover lengthening technique with 1 to 3 cm of tendon overlap. PT-to-ECRB tendon transfers were performed with native or lengthened ECRB tendons. Tensile testing was used to evaluate stress relaxation and load to failure. RESULTS: The median maximum load to failure increased with increasing overlap length, measuring 35.6 N (quartile 1, 30.2 N; quartile 3, 38.6 N) for 1 cm, 66.0 N (quartile 1, 59.1 N; quartile 3, 74.7 N) for 2 cm, and 96.6 N (quartile 1, 85.9; quartile 3, 114.9 N) for 3 cm of overlap ( P < 0.05). Failure occurred most frequently at the junction of the central overlap and native tendon. Tendons lengthened with 2 and 3 cm of overlap displayed greater stiffness than those with 1 cm ( P < 0.05). Lengthening the ECRB tendon with 2 or 3 cm of overlap did not disrupt the strength or stiffness of subsequent PT-to-ECRB tendon transfers. CONCLUSIONS: Turnover tendon lengthening does not detrimentally affect PT-to-ECRB tendon transfer. Greater overlap lengthening distance confers greater stiffness and resistance to rupture. When the periosteal extension of the PT tendon avulses or is of poor quality, the ECRB tendon can be lengthened using the turnover tendon lengthening technique to facilitate a robust transfer.

Characteristics of Upper Extremity Recovery in Acute Flaccid Myelitis: A Case Series.

BACKGROUND: Clinical characteristics and timing associated with nonsurgical recovery of upper extremity function in acute flaccid myelitis are unknown. METHODS: A single-institution retrospective case series was analyzed to describe clinical features of acute flaccid myelitis diagnosed between October of 2013 and December of 2016. Patients were consecutively sampled children with a diagnosis of acute flaccid myelitis who were referred to a hand surgeon. Patient factors and initial severity of paralysis were compared with upper extremity muscle strength outcomes using the Medical Research Council scale every 3 months up to 18 months after onset. RESULTS: Twenty-two patients with acute flaccid myelitis (aged 2 to 16 years) were studied. Proximal upper extremity musculature was more frequently and severely affected, with 56 percent of patients affected bilaterally. Functional recovery of all muscle groups (≥M3) in an individual limb was observed in 43 percent of upper extremities within 3 months. Additional complete limb recovery to greater than or equal to M3 after 3 months was rarely observed. Extraplexal paralysis, including spinal accessory (72 percent), glossopharyngeal/hypoglossal (28 percent), lower extremity (28 percent), facial (22 percent), and phrenic nerves (17 percent), was correlated with greater severity of upper extremity paralysis and decreased spontaneous recovery. There was no correlation between severity of paralysis or recovery and patient characteristics, including age, sex, comorbidities, prodromal symptoms, or time to paralysis. CONCLUSIONS: Spontaneous functional limb recovery, if present, occurred early, within 3 months of the onset of paralysis. The authors recommend that patients without signs of early recovery warrant consideration for early surgical intervention and referral to a hand surgeon or other specialist in peripheral nerve injury. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Human Fetal Scalp Dermal Papilla Enriched Genes and the Role of R-Spondin-1 in the Restoration of Hair Neogenesis in Adult Mouse Cells.

Much remains unknown about the regulatory networks which govern the dermal papilla's (DP) ability to induce hair follicle neogenesis, a capacity which decreases greatly with age. To further define the core genes which characterize the DP cell and to identify pathways prominent in DP cells with greater hair inductive capacity, comparative transcriptome analyses of human fetal and adult dermal follicular cells were performed. 121 genes were significantly upregulated in fetal DP cells in comparison to both fetal dermal sheath cup (DSC) cells and interfollicular dermal (IFD) populations. Comparison of the set of enriched human fetal DP genes with human adult DP, newborn mouse DP, and embryonic mouse dermal condensation (DC) cells revealed differences in the expression of Wnt/ß-catenin, Shh, FGF, BMP, and Notch signaling pathways. We chose R-spondin-1, a Wnt agonist, for functional verification and show that exogenous administration restores hair follicle neogenesis from adult mouse cells in skin reconstitution assays. To explore upstream regulators of fetal DP gene expression, we identified twenty-nine transcription factors which are upregulated in human fetal DP cells compared to adult DP cells. Of these, seven transcription factor binding motifs were significantly enriched in the candidate promoter regions of genes differentially expressed between fetal and adult DP cells, suggesting a potential role in the regulatory network which confers the fetal DP phenotype and a possible relationship to the induction of follicle neogenesis.

A Call to Arms: Emergency Hand and Upper-Extremity Operations During the COVID-19 Pandemic.

PURPOSE: Limited data exist regarding volumetric trends and management of upper-extremity emergencies during periods of social restriction and duress, such as the coronavirus disease 2019 pandemic. We sought to study the effect of shelter-in-place orders on emergent operative upper-extremity surgery. METHODS: All patients undergoing emergent and time-sensitive operations to the finger(s), hand, wrist, and forearm were tracked over an equal number of days before and after shelter-in-place orders at 2 geographically distinct Level I trauma centers. Surgical volume and resources, patient demographics, and injury patterns were compared before and after official shelter-in-place orders. RESULTS: A total of 58 patients underwent time-sensitive or emergent operations. Mean patient age was 42 years; mean injury severity score was 9 and median American Society of Anesthesiologist score was 2. There was a 40% increase in volume after shelter-in-place orders, averaging 1.4 cases/d. Indications for surgery included high-energy closed fracture (60%), traumatic nerve injury (19%), severe soft tissue infection (15%), and revascularization of the arm, hand, or digit(s) (15%). High-risk behavior, defined as lawlessness, assault, and high-speed auto accidents, was associated with a significantly greater proportion of operations after shelter-in-place orders (40% vs 12.5%; P < .05). Each institution dedicated an average of 3 inpatient beds and one intensive care unit-capable bed to upper-extremity care daily. Resources used included an average of 115 minutes of daily operating room time and 8 operating room staff or personnel per case. CONCLUSIONS: Hand and upper-extremity operative volume increased after shelter-in-place orders at 2 major Level I trauma centers across the country, demanding considerable hospital resources. The rise in volume was associated with an increase in high-risk behavior. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

BACKGROUND: Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. METHODS: A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. RESULTS: Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. CONCLUSION: These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Promoter choice for retroviral vectors: transcriptional strength versus trans-activation potential.

Gene expression from retroviral vectors can be driven by either the retroviral long terminal repeat (LTR) promoter or by cellular or viral promoters located internally in an LTR-deleted self-inactivating vector design. Adverse events in a gene therapy clinical trial for X-linked severe combined immune deficiency have led to the realization that the enhancer/promoter elements contained within integrated vectors may also act outside the vector genome to trans-activate host genes. Ideally, the gene expression system chosen for a vector should possess a low probability of trans-activation while still being able to support adequate levels of transgene expression. However, the parameters that define these specific characteristics are unknown. To gain insight into the mechanism of trans-activation, we compared a panel of commonly used retroviral LTRs and cellular and viral promoters for their ability to drive gene expression and to trans-activate a nearby minimal promoter in three different cell lines. These studies identified two elements, the cytomegalovirus enhancer/chicken beta-actin (CAG) and elongation factor (EF)-1alpha promoters, as being of potential value for use in vectors targeting lymphoid cells, as these elements exhibited both high levels of reporter gene expression and relatively low levels of trans-activation in T cells.

Preoperative surgical rehearsal using cadaveric fresh tissue surgical simulation increases resident operative confidence.

BACKGROUND: Rehearsal is an essential part of mastering any technical skill. The efficacy of surgical rehearsal is currently limited by low fidelity simulation models. Fresh cadaver models, however, offer maximal surgical simulation. We hypothesize that preoperative surgical rehearsal using fresh tissue surgical simulation will improve resident confidence and serve as an important adjunct to current training methods. METHODS: Preoperative rehearsal of surgical procedures was performed by plastic surgery residents using fresh cadavers in a simulated operative environment. Rehearsal was designed to mimic the clinical operation, complete with a surgical technician to assist. A retrospective, web-based survey was used to assess resident perception of pre- and post-procedure confidence, preparation, technique, speed, safety, and anatomical knowledge on a 5-point scale (1= not confident, 5= very confident). RESULTS: Twenty-six rehearsals were performed by 9 residents (PGY 1-7) an average of 4.7±2.1 days prior to performance of the scheduled operation. Surveys demonstrated a median pre-simulation confidence score of 2 and a post-rehearsal score of 4 (P<0.01). The perceived improvement in confidence and performance was greatest when simulation was performed within 3 days of the scheduled case. All residents felt that cadaveric simulation was better than standard preparation methods of self-directed reading or discussion with other surgeons. All residents believed that their technique, speed, safety, and anatomical knowledge improved as a result of simulation. CONCLUSIONS: Fresh tissue-based preoperative surgical rehearsal was effectively implemented in the residency program. Resident confidence and perception of technique improved. Survey results suggest that cadaveric simulation is beneficial for all levels of residents. We believe that implementation of preoperative surgical rehearsal is an effective adjunct to surgical training at all skill levels in the current environment of decreased work hours.

Photosensing and Thermosensing by Phytochrome B Require Both Proximal and Distal Allosteric Features within the Dimeric Photoreceptor.

Phytochromes (Phys) encompass a diverse collection of bilin-containing photoreceptors that help plants and microorganisms perceive light through photointerconversion between red light (Pr) and far-red light (Pfr)-absorbing states. In addition, Pfr reverts thermally back to Pr via a highly enthalpic process that enables temperature sensation in plants and possibly other organisms. Through domain analysis of the Arabidopsis PhyB isoform assembled recombinantly, coupled with measurements of solution size, photoconversion, and thermal reversion, we identified both proximal and distal features that influence all three metrics. Included are the downstream C-terminal histidine kinase-related domain known to promote dimerization and a conserved patch just upstream of an N-terminal Period/Arnt/Sim (PAS) domain, which upon removal dramatically accelerates thermal reversion. We also discovered that the nature of the bilin strongly influences Pfr stability. Whereas incorporation of the native bilin phytochromobilin into PhyB confers robust Pfr → Pr thermal reversion, that assembled with the cyanobacterial version phycocyanobilin, often used for optogenetics, has a dramatically stabilized Pfr state. Taken together, we conclude that Pfr acquisition and stability are impacted by a collection of opposing allosteric features that inhibit or promote photoconversion and reversion of Pfr back to Pr, thus allowing Phys to dynamically measure light, temperature, and possibly time.

Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1.

BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins.DOI: http://dx.doi.org/10.7554/eLife.02362.001.

Drug targets for rational design against emerging coronaviruses.

The recent, fatal outbreak of the novel coronavirus strain in the Middle East highlights the real threat posed by this unique virus family. Neither pharmaceutical cures nor preventive vaccines are clinically available to fight against coronavirus associated syndromes, not to mention a lack of symptom soothing drugs. Development of treatment options is complicated by the unpredictable, recurring instances of cross-species viral transmission. The vastly distributing virus reservoir and the rapid rate of host-species exchange of coronavirus demands wide spectrum potency in an ideal therapeutic. Through summarizing the available information and progress in coronavirus research, this review provides a systematic assessment of the potential wide-spectrum features on the most popular drug targets including viral proteases, spike protein, RNA polymerases and editing enzymes as well as host-virus interaction pathways associated with coronaviruses.

Recent developments on coronavirus main protease/3C like protease inhibitors.

Coronaviruses have been thrust into the spotlight by the recurring novel human coronavirus infections following the 2003 SARS pandemic. In the years since the initial SARS outbreak, the arsenal to fight this virus family has been significantly increased by the rapid growth of coronavirus research. Among the potential viral drug targets, coronavirus 3C like proteases (3Cl) have emerged as the most popular drug target. A number of patented inhibitors with promising clinical potential have been developed via different methods of drug discovery, including virtual screening, natural product isolation and structure assisted rational drug design. This review serves as a summary of the progress in both the method of drug discovery and the related inhibitors against the coronavirus 3Cl protease.

Lentiviral transduction of face and limb flaps: implications for immunomodulation of vascularized composite allografts.

BACKGROUND: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. METHODS: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. RESULTS: Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. CONCLUSIONS: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.