Current perspectives on peripheral nerve repair and management of the nerve gap.

From the first surgical repair of a nerve in the 6th century, progress in the field of peripheral nerve surgery has marched on; at first slowly but today at great pace. Whether performing primary neurorrhaphy or managing multiple large nerve defects, the modern nerve surgeon has an extensive range of tools, techniques and choices available to them. Continuous innovation in surgical equipment and technique has enabled the maturation of autografting as a gold standard for reconstruction and welcomed the era of nerve transfer techniques all while bioengineers have continued to add to our armamentarium with implantable devices, such as conduits and acellular allografts. We provide the reader a concise and up-to-date summary of the techniques available to them, and the evidence base for their use when managing nerve transection including current use and applicability of nerve transfer procedures.

A pilot prospective cohort study using experimental quantification of early peripheral nerve regeneration with high-frequency three-dimensional tomographic ultrasound (HFtUS).

Quantification of peripheral nerve regeneration after injury relies upon subjective outcome measures or electrophysiology assessments requiring fully regenerated neurons. Nerve surgeons and researchers lack objective, quantifiable information on the site of surgical repair and regenerative front. To address this need, we developed a quantifiable, visual, clinically available measure of early peripheral nerve regeneration using high-frequency, three-dimensional, tomographic ultrasound (HFtUS). We conducted a prospective, longitudinal study of adult patients with ulnar and/or median nerve injury of the arm undergoing direct epineurial repair within 5 days of injury. Assessment of morphology, volumetric and 3D grey-scale quantification of cross-sectional views were made at baseline up to 15 months post-surgery. Sensory and motor clinical outcome measures and patient reported outcome measures (PROMs) were recorded. Five participants were recruited to the study. Our data demonstrated grey-scale values (an indication of axonal density) increased in distal stumps within 2-4 months after repair, returning to normal as regeneration completed (4-6 months) with concomitant reduction in intraneural volume as surgical oedema resolved. Two patients with abnormal regeneration were characterized by increased intraneural volume and minimal grey-scale change. HFtUS may quantify early peripheral nerve regeneration offering a window of opportunity for surgical intervention where early abnormal regeneration is detected.

Treatment of Symptomatic Lymphedema in the Hand with Omental Flow-through Flap.

Vascularized lymph node transfer (VLNT) is a surgical option to improve physiologic lymphatic drainage. This technique transfers healthy vascularized lymphatic tissue from various available donor sites to the existing lymphatics of the affected area. Here, we present a successful case halting the size progression and reversing lymphedema symptoms in a patient treated with vascularized omental lymph node transfer. A 56-year-old man presented with stage III malignant sarcoma of his left medial upper arm. Two-years after excision, flap reconstruction, and radiation brachytherapy, worsening diffuse left arm edema developed, causing pain, decreased range of motion, and paresthesia. A vascularized omental lymph node transfer was performed. The omental flap required a flow-through design, requiring anastomosis of both gastroepiploic arteries to obtain Dopplerable signals. The patient experienced progressive relief of lymphedema symptoms after this transfer. Treatment outcomes with the use of VLNT have been largely encouraging; however, objective measures of improvement and timing of neolymphangiogenesis in recipient lymph node sites still need to be defined. Understanding omental VLNT flow dynamics and expected time point changes during the postoperative course will define expected outcomes and allow for treatment of a greater number of patients affected by lymphedema.

Breast-Q sensory outcomes of non-neurotized, autologous, unilateral breast reconstruction with a minimum of 3-year follow-up.

INTRODUCTION: Sensory reinnervation of autologous breast tissue after free flap reconstruction is highly variable. There is no long-term follow-up data exploring spontaneous reinnervation and how this affects patients' quality of life nor the nerve-related symptoms they experience. To address this issue, we invited patients with a minimum of 3 years after non-neurotized, free flap breast reconstruction to complete patient-reported outcome measures exploring sensation, quality of life and breast-related symptoms. METHODS: We performed a retrospective cohort study of patients undergoing unilateral Muscle-Sparing Transverse Rectus Abdominus Muscle (MS-TRAM) or deep inferior epigastric artery perforator (DIEP) flap breast reconstruction between 01-01-2015 and 31-12-2019 in the Department of Plastic and Reconstructive Surgery at Manchester University NHS Foundation Trust. We invited participants to complete the recently developed Breast-Q© Breast Sensation Module. RESULTS: All patients had undergone unilateral immediate (n = 85) or delayed (n = 82) breast reconstruction after mastectomy using either a free DIEP (n = 150) or TRAM (n = 17) flap reconstruction a minimum of 3 years prior. The median age at operation was 48. Sensation after reconstruction was significantly reduced in the reconstructed breast compared with the contralateral breast (P < 0.0001) with a reduction in reported quality of life (immediate (68.0 [54.0, 89.0]) and delayed (68.0 [62.0, 83.8])). The sensation was significantly better in immediate vs delayed procedures (P = 0.024). Sensory scores after reconstruction increased with age (P = 0.036). DISCUSSION: Breast sensation after non-neurotized reconstruction with autologous tissue is significantly reduced at long-term follow-up with a reduction in quality of life. A minimum outcome set for quantification of breast sensation is required and future research into the cost-benefit of neurotized, autologous breast reconstruction is needed.

Toward novel treatment against filariasis: Insight into genome-wide co-evolutionary analysis of filarial nematodes and Wolbachia.

Infectious diseases caused by filarial nematodes are major health problems for humans and animals globally. Current treatment using anti-helminthic drugs requires a long treatment period and is only effective against the microfilarial stage. Most species of filarial nematodes harbor a specific strain of Wolbachia bacteria, which are essential for the survival, development, and reproduction of the nematodes. This parasite-bacteria obligate symbiosis offers a new angle for the cure of filariasis. In this study, we utilized publicly available genome data and putative protein sequences from seven filarial nematode species and their symbiotic Wolbachia to screen for protein-protein interactions that could be a novel target against multiple filarial nematode species. Genome-wide in silico screening was performed to predict molecular interactions based on co-evolutionary signals. We identified over 8,000 pairs of gene families that show evidence of co-evolution based on high correlation score and low false discovery rate (FDR) between gene families and obtained a candidate list that may be keys in filarial nematode-Wolbachia interactions. Functional analysis was conducted on these top-scoring pairs, revealing biological processes related to various signaling processes, adult lifespan, developmental control, lipid and nucleotide metabolism, and RNA modification. Furthermore, network analysis of the top-scoring genes with multiple co-evolving pairs suggests candidate genes in both Wolbachia and the nematode that may play crucial roles at the center of multi-gene networks. A number of the top-scoring genes matched well to known drug targets, suggesting a promising drug-repurposing strategy that could be applicable against multiple filarial nematode species.

Identification of gametocyte-associated pir genes in the rodent malaria parasite, Plasmodium chabaudi chabaudi AS.

OBJECTIVE: To analyse the transcriptional profiles of the pir multigene family of Plasmodium chabaudi chabaudi in male and female gametocytes isolated from the blood of infected mice. RESULTS: Infected red blood cells containing female and male P. chabaudi gametocytes transcribe a distinct set of genes encoded by the multigene family pir. The overall patterns are similar to what has been observed in the close relative P. berghei, but here we show that gametocyte-associated pir genes are distinct from those involved in chronic blood-stage infection and highlight a male-associated pir gene which should be the focus of future studies.

The incidence and management of peripheral nerve injury in England (2005-2020).

Peripheral nerve injury (PNI) is a significant health problem that confers lifelong impact on those injured. Current interventions are purely surgical; however, outcomes remain poor. There is a lack of high-quality epidemiological data that is needed to identify populations involved, current healthcare demands, and ensure resources are distributed to the greatest effect, to reduce the injury burden. METHODS: Anonymized hospital episode statistical (HES) data on admitted patient care was obtained from NHS Digital for all National Health Service (NHS) patients sustaining PNI of all body regions between 2005 and 2020. Total numbers of finished consultant episodes (FCEs) or FCEs/100,000 population were used to demonstrate changes in demographic variables, anatomical locations of injury, mechanisms of injury, speciality, and main operation. RESULTS: There was a mean national incidence of 11.2 (95% CI 10.9, 11.6) events per 100,000 population per year. Males were at least twice as likely (p < 0.0001) to sustain a PNI. Upper limb nerves at or distal to the wrist were most commonly injured. Knife injuries increased (p < 0.0001), whereas glass injuries decreased (p < 0.0001). Plastic surgeons increasingly managed PNI (p = 0.002) as opposed to orthopaedic surgeons (p = 0.006) or neurosurgeons (p = 0.001). There was an increase in neurosynthesis (p = 0.022) and graft procedures (p < 0.0001) during the study period. DISCUSSION: PNI is a significant national healthcare problem predominantly affecting distal, upper limb nerves of men of working age. Injury prevention strategies, improved targeted funding and rehabilitation pathways are needed to reduce the injury burden and improve patient care.

A manually curated annotation characterises genomic features of P. falciparum lncRNAs.

BACKGROUND: Important regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum. However, due to limited research and the lack of necessary experimental tools, our understanding of their role in the malaria-causing parasite remains largely unelucidated. In this work, we address one of these limitations, the lack of an updated and improved lncRNA annotation in P. falciparum. RESULTS: We generated long-read RNA sequencing data and integrated information extracted and curated from multiple sources to manually annotate lncRNAs. We identified 1119 novel lncRNAs and validated and refined 1250 existing annotations. Utilising the collated datasets, we generated evidence-based ranking scores for each annotation and characterised the distinct genomic contexts and features of P. falciparum lncRNAs. Certain features indicated subsets with potential biological significance such as 25 lncRNAs containing multiple introns, 335 lncRNAs lacking mutations in piggyBac mutagenic studies and lncRNAs associated with specific biologic processes including two new types of lncRNAs found proximal to var genes. CONCLUSIONS: The insights and the annotation presented in this study will serve as valuable tools for researchers seeking to understand the role of lncRNAs in parasite biology through both bioinformatics and experimental approaches.

Advancing Our Understanding of the Chronically Denervated Schwann Cell: A Potential Therapeutic Target?

Outcomes for patients following major peripheral nerve injury are extremely poor. Despite advanced microsurgical techniques, the recovery of function is limited by an inherently slow rate of axonal regeneration. In particular, a time-dependent deterioration in the ability of the distal stump to support axonal growth is a major determinant to the failure of reinnervation. Schwann cells (SC) are crucial in the orchestration of nerve regeneration; their plasticity permits the adoption of a repair phenotype following nerve injury. The repair SC modulates the initial immune response, directs myelin clearance, provides neurotrophic support and remodels the distal nerve. These functions are critical for regeneration; yet the repair phenotype is unstable in the setting of chronic denervation. This phenotypic instability accounts for the deteriorating regenerative support offered by the distal nerve stump. Over the past 10 years, our understanding of the cellular machinery behind this repair phenotype, in particular the role of c-Jun, has increased exponentially, creating opportunities for therapeutic intervention. This review will cover the activation of the repair phenotype in SC, the effects of chronic denervation on SC and current strategies to 'hack' these cellular pathways toward supporting more prolonged periods of neural regeneration.

Highly Variable Expression of Merozoite Surface Protein MSPDBL2 in Diverse Plasmodium falciparum Clinical Isolates and Transcriptome Scans for Correlating Genes.

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 96 clinical isolates from 4 populations with various levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2, but the frequency distribution was highly skewed, as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether the expression of other gene loci correlated with MSPDBL2 expression, whole-transcriptome sequencing was performed on schizont-enriched material from 17 of the isolates with a wide range of proportions of schizonts positive. Transcripts of particular genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, showing overrepresentation of genes implicated previously as involved in gametocytogenesis but not including the gametocytogenesis master regulator ap2-g. Single-cell transcriptome analysis of a laboratory-adapted clone showed that most individual parasites expressing mspdbl2 did not express ap2-g, consistent with MSPDBL2 marking a developmental subpopulation that is distinct but likely to co-occur alongside sexual commitment. IMPORTANCE These findings contribute to understanding malaria parasite antigenic and developmental variation, focusing on the merozoite surface protein encoded by the single locus under strongest balancing selection. Analyzing the initial ex vivo generation of parasites grown from a wide sample of clinical infections, we show a unique and highly skewed pattern of natural expression frequencies of MSPDBL2, distinct from that of any other antigen. Bulk transcriptome analysis of a range of clinical isolates showed significant overrepresentation of sexual development genes among those positively correlated with MSPDBL2 protein and mspdbl2 gene expression, indicating the MSPDBL2-positive subpopulation to be often coincident with parasites developing sexually in preparation for transmission. Single-cell transcriptome data confirm the absence of a direct correlation with the ap2-g master regulator of sexual development, indicating that the MSPDBL2-positive subpopulation has a separate function in asexual survival and replication under conditions that promote terminal sexual differentiation.

Defining the early stages of intestinal colonisation by whipworms.

Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche.

Association of socioeconomic status with 30- and 90-day readmission following open and laparoscopic hernia repair: a nationwide readmissions database analysis.

BACKGROUND: Socioeconomic disparities have been associated with outcomes in many medical conditions. The association of socioeconomic status (SES) with readmissions after ventral and inguinal hernia repair has not been well studied on a national level. This study aims to evaluate the association of SES with readmission as a significant outcome in patients undergoing ventral and inguinal hernia repair. METHODS: A retrospective cohort study was performed evaluating patients undergoing ventral hernia and inguinal hernia repair with 1:1 propensity score matching using the Nationwide Readmissions Database (2016-2017). Both 30- and 90-day readmissions were examined. After matching, a multivariate logistic regression analysis was performed using confounding variables including hospital setting, comorbidities, urgency of repair, sociodemographic status, and payer. Likelihood of readmission was reported in odds ratio form. RESULTS: Readmission rates were 11.56% (24,323 out of 210,381) and 17.94% (30,893 out of 172,210) for 30- and 90-day readmissions, respectively. Patients with Medicaid and in the lower income quartile were more likely to present in an emergent fashion for hernia repair. After matching, a multivariate logistic regression analysis showed socioeconomic status (OR 1.250 and 1.229) was a statistically significant independent predictor of readmission at 30 and 90 days, respectively. Inversely, factors associated with the least likely chance of readmission were a laparoscopic approach (OR 0.646 and 0.641), elective admission (OR 0.824 and 0.779), and care in a teaching hospital (OR 0.784 and 0.798). CONCLUSION: SES is an independent predictor of readmission at 30 and 90 days following open and laparoscopic ventral and inguinal hernia repair. Patients with a lower socioeconomic status were more likely to undergo hernia repair in the emergent setting. Efforts toward mitigating SES disparities by potentially promoting MIS techniques, enhancing access to elective cases, and systematic approaches to perioperative care for this disadvantaged population can potentially enhance overall hernia outcomes.

Emerging Roles of Cholinergic Receptors in Schwann Cell Development and Plasticity.

The cross talk between neurons and glial cells during development, adulthood, and disease, has been extensively documented. Among the molecules mediating these interactions, neurotransmitters play a relevant role both in myelinating and non-myelinating glial cells, thus resulting as additional candidates regulating the development and physiology of the glial cells. In this review, we summarise the contribution of the main neurotransmitter receptors in the regulation of the morphogenetic events of glial cells, with particular attention paid to the role of acetylcholine receptors in Schwann cell physiology. In particular, the M2 muscarinic receptor influences Schwann cell phenotype and the α7 nicotinic receptor is emerging as influential in the modulation of peripheral nerve regeneration and inflammation. This new evidence significantly improves our knowledge of Schwann cell development and function and may contribute to identifying interesting new targets to support the activity of these cells in pathological conditions.

Daily rhythms in gene expression of the human parasite Schistosoma mansoni.

BACKGROUND: The consequences of the earth's daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis. RESULTS: Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host's active phase and a 'peak in metabolic activity' during the host's resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity. CONCLUSIONS: There are daily rhythms in the transcriptomes of adult S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis.

Analysis of pir gene expression across the Plasmodium life cycle.

BACKGROUND: Plasmodium interspersed repeat (pir) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection. METHODS: Here pir gene expression was examined across the life cycle of Plasmodium berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from Plasmodium chabaudi. RESULTS: Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology. CONCLUSIONS: The identification of distinct expression patterns for different pir genes and subfamilies is likely to provide a basis for the design of future experiments to uncover their function.

Controlled human malaria infection with a clone of Plasmodium vivax with high-quality genome assembly.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, 2 healthy malaria-naive United Kingdom adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers, and prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected RBCs. Following stringent safety screening, the parasite stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-naive United Kingdom adults by blood-stage CHMI, at 3 different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high-quality genome assembly by using a hybrid assembly method. We analyzed leading vaccine candidate antigens and multigene families, including the vivax interspersed repeat (VIR) genes, of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.

Transcriptomic Profile Reveals Deregulation of Hearing-Loss Related Genes in Vestibular Schwannoma Cells Following Electromagnetic Field Exposure.

Hearing loss (HL) is the most common sensory disorder in the world population. One common cause of HL is the presence of vestibular schwannoma (VS), a benign tumor of the VIII cranial nerve, arising from Schwann cell (SC) transformation. In the last decade, the increasing incidence of VS has been correlated to electromagnetic field (EMF) exposure, which might be considered a pathogenic cause of VS development and HL. Here, we explore the molecular mechanisms underlying the biologic changes of human SCs and/or their oncogenic transformation following EMF exposure. Through NGS technology and RNA-Seq transcriptomic analysis, we investigated the genomic profile and the differential display of HL-related genes after chronic EMF. We found that chronic EMF exposure modified the cell proliferation, in parallel with intracellular signaling and metabolic pathways changes, mostly related to translation and mitochondrial activities. Importantly, the expression of HL-related genes such as NEFL, TPRN, OTOGL, GJB2, and REST appeared to be deregulated in chronic EMF exposure. In conclusion, we suggest that, at a preclinical stage, EMF exposure might promote the transformation of VS cells and contribute to HL.

A single-cell atlas of Plasmodium falciparum transmission through the mosquito.

Malaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria - Plasmodium falciparum. We reveal developmental trajectories associated with the colonization of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and non-conserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, provide a fine-grained atlas that enables intensive investigation of the P. falciparum transcriptional journey. As well as providing insights into gene function across the transmission cycle, the atlas opens the door for identification of drug and vaccine targets to stop malaria transmission and thereby prevent disease.