Effect of knocking out mouse Slc44a4 on colonic uptake of the microbiota-generated thiamine pyrophosphate and colon physiology.

Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation.NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.

Changes in the innate immune response to SARS-CoV-2 with advancing age in humans.

BACKGROUND: Advancing age is a major risk factor for respiratory viral infections. The infections are often prolonged and difficult to resolve resulting hospitalizations and mortality. The recent COVID-19 pandemic has highlighted this as elderly subjects have emerged as vulnerable populations that display increased susceptibility and severity to SARS-CoV-2. There is an urgent need to identify the probable mechanisms underlying this to protect against future outbreaks of such nature. Innate immunity is the first line of defense against viruses and its decline impacts downstream immune responses. This is because dendritic cells (DCs) and macrophages are key cellular elements of the innate immune system that can sense and respond to viruses by producing inflammatory mediators and priming CD4 and CD8 T-cell responses. RESULTS: We investigated the changes in innate immune responses to SARS-CoV-2 as a function of age. Our results using human PBMCs from aged, middle-aged, and young subjects indicate that the activation of DCs and monocytes in response to SARS-CoV-2 is compromised with age. The impairment is most apparent in pDCs where both aged and middle-aged display reduced responses. The secretion of IL-29 that confers protection against respiratory viruses is also decreased in both aged and middle-aged subjects. In contrast, inflammatory mediators associated with severe COVID-19 including CXCL-8, TREM-1 are increased with age. This is also apparent in the gene expression data where pathways related host defense display an age dependent decrease with a concomitant increase in inflammatory pathways. Not only are the inflammatory pathways and mediators increased after stimulation with SARS-CoV-2 but also at homeostasis. In keeping with reduced DC activation, the induction of cytotoxic CD8 T cells is also impaired in aged subjects. However, the CD8 T cells from aged subjects display increased baseline activation in accordance with the enhanced baseline inflammation. CONCLUSIONS: Our results demonstrate a decline in protective anti-viral immune responses and increase in damaging inflammatory responses with age indicating that dysregulated innate immune responses play a significant role in the increased susceptibility of aged subjects to COVID-19. Furthermore, the dysregulation in immune responses develops early on as middle-aged demonstrate several of these changes.

Bacterial Organisms Colonizing Mohs Surgical Wounds Healing by Second Intention on the Lower Extremities: A Prospective, Controlled Study.

BACKGROUND: Based on recent research, second intention wounds may be at greater risk for postoperative infection compared with sutured wounds. Accurate diagnosis of postoperative wound infections on the lower leg can be challenging and result in unnecessary antibiotic administration. OBJECTIVE: The objective of this study was to identify bacterial organisms that commonly colonize second intention surgical wounds after Mohs micrographic surgery (MMS) and isolate pathogenic organisms. METHODS: Patients with second intention surgical wounds on the lower leg were evaluated 2 weeks after MMS. Wounds were swabbed for bacteria and categorized as infected or not infected based on clinical appearance. Any colonizing bacteria were recorded once the culture results were reported. RESULTS: Twenty-five clinically infected wounds and 26 control wounds were cultured. Staphylococcus aureus was the most common bacteria, colonizing 8 infected wounds (15.7%) and 5 control wounds (9.8%). Eight cultures (32%) from clinically infected wounds grew normal skin flora alone. CONCLUSION: Staphylococcus aureus is the most common bacteria colonizing wounds healing by second intention on the lower extremity. Thirty-two percent of clinically infected wounds grew normal skin flora, demonstrating the challenge of accurately diagnosing infection in lower extremity second intention wounds.

Procedural and surgical treatment modalities for acne scarring - Part 2.

The optimal treatment of acne scarring is challenging because it involves consideration of several factors, including the type and number of scars, Fitzpatrick skin type, and the amount of downtime permissible to the patient. The second article in this CME series discusses the procedural treatments available for acne scarring, including the use of chemical peels, fillers, radiofrequency microneedling, lasers and surgical procedures. The indications for each modality, evidence for its benefits, and the adverse effects are discussed. This section aims to help guide the reader to select and implement the most appropriate treatment based on the patient's preferences, acne scar and skin type.

Acne scarring-pathophysiology, diagnosis, prevention and education - Part 1.

Acne scarring is common and can occur despite effective acne management. Acne scarring patients suffer from significant psychosocial morbidity including depression and suicidality. Despite availability and advancement of therapeutic modalities, treatment for acne scarring is uncommon and often overlooked in the acne patient encounter. The utilization of acne scarring assessment tools and identification of specific acne scar subtypes allow for a tailored therapeutic approach. Part I of this continuing medical education series covers the pathophysiology and morphology of textural and pigmented acne scars, scarring assessment tools and medical treatment options. The principles reviewed will aid in the approach and initiation of acne scar treatment in the outpatient setting.

Recurrence Rate for Melanoma Excised by Mohs Micrographic Surgery Without Immunostaining.

BACKGROUND: Mohs micrographic surgery (MMS) is becoming increasingly popular for the treatment of cutaneous melanoma (CM) as multiple studies have demonstrated favorable outcomes for local recurrence and overall survival. OBJECTIVE: To analyze the outcomes of noninvasive (NIM) and invasive melanomas (IM) using MMS with fresh frozen sections. The primary outcome was local recurrence. The secondary outcome was to identify mean surgical margins based on tumor type and location. METHODS: Retrospective cohort study of 224 cases of CM treated from 2006 to 2016 at a tertiary academic center with MMS and fresh frozen sections by a single Mohs surgeon. RESULTS: The overall recurrence rate was 2.6% with a mean follow-up of 36.2 months. The recurrence rate for NIM versus IM was 1.6% and 7%, respectively. The mean margins for NIM and IM were 7.9 mm and 10.1 mm, respectively. These varied by tumor site and location. CONCLUSION: This study supports the use of MMS in the treatment of CM and highlights how narrower surgical margins for NIM of the head and neck can be achieved without increasing risk of local recurrence.

Intravenous immunoglobulin G use in patients with chronic inflammatory demyelinating polyneuropathy: An uncommon cause of drug-induced discoid lupus erythematosus.

Drug-induced chronic cutaneous lupus erythematosus, or drug-induced discoid lupus erythematosus, is a rare cutaneous phenomenon. Various medications have been associated with drug-induced discoid lupus erythematosus including fluorouracile agents, especially tegafur and uraciltegafur, and TNF-α antagonists such as infliximab or etanercept. Recent literature has described a case series of six patients receiving IgG immunoglobulin for chronic inflammatory demyelinating polyneuropathy with subsequent presentations of discoid lupus erythematosus. We present a patient with chronic inflammatory demyelinating polyneuropathy who developed discoid lupus erythematosus secondary to IgG immunoglobulin.

Endothelium-Independent Primitive Myxoid Vascularization Creates Invertebrate-Like Channels to Maintain Blood Supply in Optic Gliomas.

Optic gliomas are brain tumors characterized by slow growth, progressive loss of vision, and limited therapeutic options. Optic gliomas contain various amounts of myxoid matrix, which can represent most of the tumor mass. We sought to investigate biological function and protein structure of the myxoid matrix in optic gliomas to identify novel therapeutic targets. We reviewed histological features and clinical imaging properties, analyzed vasculature by immunohistochemistry and electron microscopy, and performed liquid chromatography-mass spectrometry on optic gliomas, which varied in the amount of myxoid matrix. We found that although subtypes of optic gliomas are indistinguishable on imaging, the microvascular network of pilomyxoid astrocytoma, a subtype of optic glioma with abundant myxoid matrix, is characterized by the presence of endothelium-free channels in the myxoid matrix. These tumors show normal perfusion by clinical imaging and lack histological evidence of hemorrhage organization or thrombosis. The myxoid matrix is composed predominantly of the proteoglycan versican and its linking protein, a vertebrate hyaluronan and proteoglycan link protein 1. We propose that pediatric optic gliomas can maintain blood supply without endothelial cells by using invertebrate-like channels, which we termed primitive myxoid vascularization. Enzymatic targeting of the proteoglycan versican/hyaluronan and proteoglycan link protein 1 rich myxoid matrix, which is in direct contact with circulating blood, can provide novel therapeutic avenues for optic gliomas of childhood.

Acquired Lymphangiectasia in the Setting of a Gastrointestinal Stromal Tumor.

A 53-year-old woman presented for a skin cancer screening and was found to have scattered violaceous papulovesicles on the right buttock and right lower aspect of the abdomen. These lesions had been increasing in number for 3 years. The patient denied pain or pruritis at the site of lesions and denied any systemic fi ndings.

Extracting seizure frequency from epilepsy clinic notes: a machine reading approach to natural language processing.

OBJECTIVE: Seizure frequency and seizure freedom are among the most important outcome measures for patients with epilepsy. In this study, we aimed to automatically extract this clinical information from unstructured text in clinical notes. If successful, this could improve clinical decision-making in epilepsy patients and allow for rapid, large-scale retrospective research. MATERIALS AND METHODS: We developed a finetuning pipeline for pretrained neural models to classify patients as being seizure-free and to extract text containing their seizure frequency and date of last seizure from clinical notes. We annotated 1000 notes for use as training and testing data and determined how well 3 pretrained neural models, BERT, RoBERTa, and Bio_ClinicalBERT, could identify and extract the desired information after finetuning. RESULTS: The finetuned models (BERTFT, Bio_ClinicalBERTFT, and RoBERTaFT) achieved near-human performance when classifying patients as seizure free, with BERTFT and Bio_ClinicalBERTFT achieving accuracy scores over 80%. All 3 models also achieved human performance when extracting seizure frequency and date of last seizure, with overall F1 scores over 0.80. The best combination of models was Bio_ClinicalBERTFT for classification, and RoBERTaFT for text extraction. Most of the gains in performance due to finetuning required roughly 70 annotated notes. DISCUSSION AND CONCLUSION: Our novel machine reading approach to extracting important clinical outcomes performed at or near human performance on several tasks. This approach opens new possibilities to support clinical practice and conduct large-scale retrospective clinical research. Future studies can use our finetuning pipeline with minimal training annotations to answer new clinical questions.

Microsatellite primers for the Pacific Northwest endemic conifer Chamaecyparis lawsoniana (Cupressaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the Pacific Northwest conifer, Chamaecyparis lawsoniana (Cupressaceae), to enhance efficiencies in disease-resistance breeding and germplasm screening for gene conservation of this rare species. METHODS AND RESULTS: Using multiplexed massively parallel Illumina sequencing, we identified over 300000 microsatellite-containing sequences from 2 million paired-end microreads. After stringent filtering and primer evaluation, we selected 11 primer pairs and used these to screen variation in four populations of C. lawsoniana. Loci show between three and 10 repeats per locus, with an average of eight. Screening of these markers in the North American relative Callitropsis nootkatensis demonstrated limited marker transferability, but these markers could have utility in Asian species of Chamaecyparis. CONCLUSIONS: These microsatellite primers show high polymorphism and should provide a high level of individual discrimination for paternity analysis in defined pedigrees, and routine screening of wild variation in Chamaecyparis lawsoniana.

Responsive neurostimulation targeting anterior thalamic nucleus in generalized epilepsy.

Responsive neurostimulation (RNS) has emerged as an adjunctive treatment modality for patients with intractable focal epilepsy who are not surgical candidates or have more than one ictal onset focus. We report a 34-year-old patient with intractable, childhood-onset, genetic generalized epilepsy (GGE) with tonic, atonic, myoclonic and absence seizures treated with RNS. Strip electrodes over the right posterior frontal cortex and depth electrodes placed in the right anterior nucleus were used for event detection and responsive stimulation. Two-year follow-up revealed 90-95% clinical seizure reduction. This case suggests that refractory GGE may be effectively treated with RNS targeting thalamocortical networks.

Special Considerations in the Treatment of Mycosis Fungoides.

Mycosis fungoides is the most common form of cutaneous T cell lymphoma. Although normally presenting to physicians at an early stage and with an indolent course, mycosis fungoides can have a varied presentation. The National Comprehensive Cancer Network (NCCN) has created guidelines for the treatment and staging of mycosis fungoides. Although comprehensive, in practice these guidelines do not provide specific treatment regimens for lesions located in difficult locations and those recalcitrant to the recommended therapy. Because of this, suggestions based on the practices and decisions made at the multidisciplinary cutaneous lymphoma clinic at the Sidney Kimmel Cancer Center at Thomas Jefferson University, Philadelphia, PA, USA, are presented here. Lesions located in areas such as the face and intertriginous zones are often challenging to treat because first-line therapies are often inappropriate, with the locations increasing the possibility of side effects. Additionally, lesions located in the bathing suit distribution are often nonresponsive to first-line therapies for reasons still undetermined. Finally, although well-described, erythroderma secondary to mycosis fungoides is challenging to treat, with controversy surrounding various methods of control. This article both highlights difficult clinical scenarios and reviews the recommended treatment as provided by the NCCN guidelines and provides alternative therapy for lesions that are either difficult to treat because of the location or are recalcitrant to the recommended therapy. With suggestions for the apparent gaps in guidelines, providers can better treat patients who present with more complicated conditions.

Valchlor maintenance therapy for patients with mycosis fungoides who received low dose total skin electron beam treatment.

Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma within the general population. Low dose total skin electron beam therapy (TSEBT) and topical nitrogen mustard (Valchlor) are two treatment modalities that have been proven to be efficacious in the treatment of MF. While each have been studied independently in various clinical trials, the use of Valchlor as maintenance therapy after completion of low dose TSEBT is rarely implemented due to the lack of evidence in the literature. The Jefferson multidisciplinary cutaneous lymphoma clinic has found great success with this combination of treatment and it was the goal of the authors to provide further evidence to its efficacy. The authors conducted a retrospective review of eight patients at the Jefferson multidisciplinary cutaneous lymphoma clinic period. In this study, they were initiated on a regimen of Valchlor as a maintenance therapy after completion of low dose TSEBT. The median MSWAT score before low dose TSEBT was found to be 25.25 with a mean of 39.76. A reduction was found in MSWAT score after low dose TSEBT to a median of 7.68 and a mean of 17.31. Median scores for pruritus were decreased from 3.43 before TSEBT to 1.88 after low dose TSEBT and a decreased in quality of life score median from 6.60 to a median of 2.75. Valchlor proved to be a useful maintenance therapy prolonging time to stage increase by 22.7351 months. Overall this study provides further evidence to the efficacy of Valchlor used as a maintenance therapy after completion of low dose TSEBT.

Long-term clinical and visual outcomes after surgical resection of pediatric pilocytic/pilomyxoid optic pathway gliomas.

OBJECTIVE: The choice of treatment modality for optic pathway gliomas (OPGs) is controversial. Chemotherapy is widely regarded as first-line therapy; however, subtotal resections have been reported for decompression or salvage therapy as first- and second-line treatment. The goal of this study was to further investigate the role and efficacy of resection for OPGs. METHODS: A retrospective chart review was performed on 83 children who underwent surgical treatment for OPGs between 1986 and 2014. Pathology was reviewed by a neuropathologist. Clinical outcomes, including progression-free survival (PFS), overall survival (OS), and complications, were analyzed. RESULTS: The 5- and 10-year PFS rates were 55% and 46%, respectively. The 5- and 10-year OS rates were 87% and 78%, respectively. The median extent of resection was 80% (range 30%-98%). Age less than 2 years at surgery and pilomyxoid features of the tumor were found to be associated with significantly lower 5-year OS. No difference was seen in PFS or OS of children treated with surgery as a first-line treatment compared with children with surgery as a second- or third-line treatment. Severe complications included new disabling visual deficit in 5%, focal neurological deficit in 8%, and infection in 2%. New hormone deficiency occurred in 22% of the children. CONCLUSIONS: Approximately half of all children experience a long-term benefit from resection both as primary treatment and as a second-line therapy after failure of primary treatment. Primary surgery does not appear to have a significant benefit for children younger than 2 years or tumors with pilomyxoid features. Given the risks associated with surgery, an interdisciplinary approach is needed to tailor the treatment plan to the individual characteristics of each child.

The histological prevalence and clinical implications of folliculotropism and syringotropism in mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma, remains a challenge for clinicians to stage and manage. Classically, MF is determined through histopathologic evidence of a neoplastic infiltrate within the epidermis. In certain patients, however, the infiltrate extends into the hair follicles and sweat glands. The objective of this study is to determine the utility of expanding the analysis of histopathology reports to include the reporting of folliculotropism and syringotropism. METHODS: This is a prospective, observational study conducted in a single facility from 2012-2018. All patients with MF, excluding those treated at this facility for less than six months with the exception of those with incomplete pruritus documentation, or absence of initial biopsy analysis were studied. Modified severity weighted assessment tool (mSWAT) quantified body surface area and treatments attempted per patient were continuously charted. Patients were surveyed for presence and degree of pruritus and pain. Evaluation of these parameters were charted at the initial patient visit and correlated with their primary biopsy for presence or absence of folliculotropism and syringotropism. RESULTS: Of the 87 patients examined, 70 patients (80%) exhibited syringotropism in their original biopsy and 68 patients (78%) exhibited folliculotropism. Presence of both findings concurrently was present in 56 patients (64.4%), while neither finding was present in 5 patients (5.8%). The singular finding of folliculotropism was found in 12 patients (13.8%), while the singular finding of syringotropism was exhibited in 14 patients (16.1%). A significant association between the presence of folliculotropism and pruritus was established (P=0.043, α=0.05). The general trend towards increase in mSWAT score and pruritus in patients in regard to the mean and median values suggest that increasing the sample population of the study might yield a significant value in the future. CONCLUSIONS: These presentations are more prevalent than previously recognized and have findings indicative of more severe disease. We propose that MF histopathology reports document the presence of folliculotropism and syringotropism and that these findings be added to the NCCN guidelines as they may aid in predicting severity and progression risk.

Development of nuclear microsatellite loci for Pinus albicaulis Engelm. (Pinaceae), a conifer of conservation concern.

Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel's tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.

Development of Molecular Markers for Determining Continental Origin of Wood from White Oaks (Quercus L. sect. Quercus).

To detect and avoid illegal logging of valuable tree species, identification methods for the origin of timber are necessary. We used next-generation sequencing to identify chloroplast genome regions that differentiate the origin of white oaks from the three continents; Asia, Europe, and North America. By using the chloroplast genome of Asian Q. mongolica as a reference, we identified 861 variant sites (672 single nucleotide polymorphisms (SNPs); 189 insertion/deletion (indel) polymorphism) from representative species of three continents (Q. mongolica from Asia; Q. petraea and Q. robur from Europe; Q. alba from North America), and we identified additional chloroplast polymorphisms in pools of 20 individuals each from Q. mongolica (789 variant sites) and Q. robur (346 variant sites). Genome sequences were screened for indels to develop markers that identify continental origin of oak species, and that can be easily evaluated using a variety of detection methods. We identified five indels and one SNP that reliably identify continent-of-origin, based on evaluations of up to 1078 individuals representing 13 white oak species and three continents. Due to the size of length polymorphisms revealed, this marker set can be visualized using capillary electrophoresis or high resolution gel (acrylamide or agarose) electrophoresis. With these markers, we provide the wood trading market with an instrument to comply with the U.S. and European laws that require timber companies to avoid the trade of illegally harvested timber.

Targeted Capture Sequencing in Whitebark Pine Reveals Range-Wide Demographic and Adaptive Patterns Despite Challenges of a Large, Repetitive Genome.

Whitebark pine (Pinus albicaulis) inhabits an expansive range in western North America, and it is a keystone species of subalpine environments. Whitebark is susceptible to multiple threats - climate change, white pine blister rust, mountain pine beetle, and fire exclusion - and it is suffering significant mortality range-wide, prompting the tree to be listed as 'globally endangered' by the International Union for Conservation of Nature and 'endangered' by the Canadian government. Conservation collections (in situ and ex situ) are being initiated to preserve the genetic legacy of the species. Reliable, transferrable, and highly variable genetic markers are essential for quantifying the genetic profiles of seed collections relative to natural stands, and ensuring the completeness of conservation collections. We evaluated the use of hybridization-based target capture to enrich specific genomic regions from the 27 GB genome of whitebark pine, and to evaluate genetic variation across loci, trees, and geography. Probes were designed to capture 7,849 distinct genes, and screening was performed on 48 trees. Despite the inclusion of repetitive elements in the probe pool, the resulting dataset provided information on 4,452 genes and 32% of targeted positions (528,873 bp), and we were able to identify 12,390 segregating sites from 47 trees. Variations reveal strong geographic trends in heterozygosity and allelic richness, with trees from the southern Cascade and Sierra Range showing the greatest distinctiveness and differentiation. Our results show that even under non-optimal conditions (low enrichment efficiency; inclusion of repetitive elements in baits), targeted enrichment produces high quality, codominant genotypes from large genomes. The resulting data can be readily integrated into management and gene conservation activities for whitebark pine, and have the potential to be applied to other members of 5-needle pine group (Pinus subsect. Quinquefolia) due to their limited genetic divergence.