The status of the human gene catalogue.

Scientists have been trying to identify every gene in the human genome since the initial draft was published in 2001. In the years since, much progress has been made in identifying protein-coding genes, currently estimated to number fewer than 20,000, with an ever-expanding number of distinct protein-coding isoforms. Here we review the status of the human gene catalogue and the efforts to complete it in recent years. Beside the ongoing annotation of protein-coding genes, their isoforms and pseudogenes, the invention of high-throughput RNA sequencing and other technological breakthroughs have led to a rapid growth in the number of reported non-coding RNA genes. For most of these non-coding RNAs, the functional relevance is currently unclear; we look at recent advances that offer paths forward to identifying their functions and towards eventually completing the human gene catalogue. Finally, we examine the need for a universal annotation standard that includes all medically significant genes and maintains their relationships with different reference genomes for the use of the human gene catalogue in clinical settings.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Effects of transcriptional noise on estimates of gene and transcript expression in RNA sequencing experiments.

RNA sequencing is widely used to measure gene expression across a vast range of animal and plant tissues and conditions. Most studies of computational methods for gene expression analysis use simulated data to evaluate the accuracy of these methods. These simulations typically include reads generated from known genes at varying levels of expression. Until now, simulations did not include reads from noisy transcripts, which might include erroneous transcription, erroneous splicing, and other processes that affect transcription in living cells. Here we examine the effects of realistic amounts of transcriptional noise on the ability of leading computational methods to assemble and quantify the genes and transcripts in an RNA sequencing experiment. We show that the inclusion of noise leads to systematic errors in the ability of these programs to measure expression, including systematic underestimates of transcript abundance levels and large increases in the number of false-positive genes and transcripts. Our results also suggest that alignment-free computational methods sometimes fail to detect transcripts expressed at relatively low levels.

Paragangliomas of the Head and Neck: A Review of the Latest Diagnostic and Treatment Methods.

Background and objectives: Paragangliomas of the head and neck are rare, slow-growing neuroendocrine tumors, benign in their vast majority, but with a possibility of developing distant metastases. They show great inheritable character, and their behavior has proven to be unpredictable; therefore, they are considered malignant. Material and methods: This article aims to offer a more comprehensive presentation of the pathogenesis, epidemiology, diagnostic methods, imaging development, and treatment guidelines. We tried to bring together all the necessary data that, in our opinion, a head and neck practitioner should know when managing this type of tumor. Our main focus is on the most recent studies, with the purpose of a homogenous presentation of all current guidelines and approaches to this pathology. Results: Paragangliomas of the head and neck are still a disputed topic. One of the main reasons for that is their low incidence of 0.3 to 1 per 100,000 every year. The most frequent locations are the carotid body, the temporal bone, the jugular and mastoid foramen, and the vagal nerve. Their clinical presentation usually involves a painless lateral mass associated with symptoms such as hoarseness, hearing loss, tinnitus, and cranial nerve deficits. Up to 40% of them are inherited, mostly linked with mutations of succinate dehydrogenase complex. Imaging evaluation consists of CT and MRI, and new functional explorations such as 18F-FDA and 18F-FDG PET/CT, 18F-DOPA PET, 123I-MIBG, and 68Ga-DOTATE PET/CT. Measuring the catecholamine levels in the plasma and urine is mandatory, even though paragangliomas of the head and neck rarely display secretory behavior. Treatment mainly consists of surgery, with different approaches and techniques, but conservative management methods such as wait and scan, radiotherapy, proton therapy, and chemotherapy have proven their efficiency. The therapeutical decision lacks consensus, and current studies tend to recommend an individualized approach. Guidelines regarding long-term follow-up are still a matter of debate.

Improved transcriptome assembly using a hybrid of long and short reads with StringTie.

Short-read RNA sequencing and long-read RNA sequencing each have their strengths and weaknesses for transcriptome assembly. While short reads are highly accurate, they are rarely able to span multiple exons. Long-read technology can capture full-length transcripts, but its relatively high error rate often leads to mis-identified splice sites. Here we present a new release of StringTie that performs hybrid-read assembly. By taking advantage of the strengths of both long and short reads, hybrid-read assembly with StringTie is more accurate than long-read only or short-read only assembly, and on some datasets it can more than double the number of correctly assembled transcripts, while obtaining substantially higher precision than the long-read data assembly alone. Here we demonstrate the improved accuracy on simulated data and real data from Arabidopsis thaliana, Mus musculus, and human. We also show that hybrid-read assembly is more accurate than correcting long reads prior to assembly while also being substantially faster. StringTie is freely available as open source software at https://github.com/gpertea/stringtie.

Polymicrobial Bacterial Meningitis in a Patient with Chronic Suppurative Otitis Media: Case Report and Literature Review.

Polymicrobial meningitis is a rare entity in the adult population, especially in the antibiotic era. However, disorders such as chronic suppurative otitis media (CSOM) or even poor oral hygiene are considered risk factors for the development of such cerebral infection. We report a case of polymicrobial meningitis associated with oto-mastoiditis in a 64-year-old female patient known to have CSOM. The patient presented atypical symptoms for community-acquired meningitis, showing subacute evolution of headache, without fever or neck stiffness. The aerobe microorganisms Streptococcus anginosus and Corynebacterium spp., sensitive to beta-lactamines, and the anaerobe Prevotella spp., resistant to penicillin and metronidazole, were isolated from CSF specimens, while Proteus mirabilis and Enterococcus faecalis were identified from the ear drainage. The diversity of pathogens identified in our case led us to the hypothesis of two different sources of meningitis: otogenic and/or odontogenic. Favorable evolution was obtained after a multi-disciplinary approach, combining surgery and broad-spectrum antibiotics. In addition, we performed a literature review that highlights the low incidence of polymicrobial mixed aerobe-anaerobe meningitis.

Human contamination in bacterial genomes has created thousands of spurious proteins.

Contaminant sequences that appear in published genomes can cause numerous problems for downstream analyses, particularly for evolutionary studies and metagenomics projects. Our large-scale scan of complete and draft bacterial and archaeal genomes in the NCBI RefSeq database reveals that 2250 genomes are contaminated by human sequence. The contaminant sequences derive primarily from high-copy human repeat regions, which themselves are not adequately represented in the current human reference genome, GRCh38. The absence of the sequences from the human assembly offers a likely explanation for their presence in bacterial assemblies. In some cases, the contaminating contigs have been erroneously annotated as containing protein-coding sequences, which over time have propagated to create spurious protein "families" across multiple prokaryotic and eukaryotic genomes. As a result, 3437 spurious protein entries are currently present in the widely used nr and TrEMBL protein databases. We report here an extensive list of contaminant sequences in bacterial genome assemblies and the proteins associated with them. We found that nearly all contaminants occurred in small contigs in draft genomes, which suggests that filtering out small contigs from draft genome assemblies may mitigate the issue of contamination while still keeping nearly all of the genuine genomic sequences.

TieBrush: an efficient method for aggregating and summarizing mapped reads across large datasets.

SUMMARY: Although the ability to programmatically summarize and visually inspect sequencing data is an integral part of genome analysis, currently available methods are not capable of handling large numbers of samples. In particular, making a visual comparison of transcriptional landscapes between two sets of thousands of RNA-seq samples is limited by available computational resources, which can be overwhelmed due to the sheer size of the data. In this work, we present TieBrush, a software package designed to process very large sequencing datasets (RNA, whole-genome, exome, etc.) into a form that enables quick visual and computational inspection. TieBrush can also be used as a method for aggregating data for downstream computational analysis, and is compatible with most software tools that take aligned reads as input. AVAILABILITY AND IMPLEMENTATION: TieBrush is provided as a C++ package under the MIT License. Precompiled binaries, source code and example data are available on GitHub (https://github.com/alevar/tiebrush). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pain Intensity and Degree of Disability after Fragility Fractures of the Pelvis.

Background and objectives: Fragility fractures of the pelvis (FFP) are of increasing interest lately, being associated with a loss of mobility and affecting the quality of life. The aim of our study was to investigate the effect of FFP on disability and pain in patients, after one year since injury. Materials and Methods: In the study, we included 76 patients diagnosed with FFP, who were admitted to our trauma department between January 2016 and January 2019, and were above 65 years of age. The Von Korff pain intensity and disability scores were calculated in the hospital at 6 months and after 1 year. Results: Fifty-four patients were female (71%), with an average age of 75.9 ± 7.19 years. Twenty-two patients were male (29%) and had a mean age of 77.22 ± 7.33 years. We did not record significant differences regarding age between the men and women (p > 0.05). Significant improvements appeared between the baseline and the 6 month follow-up; the average pain intensity score at 6 months was 44.94 (SD 21.20) (p < 0.001), and the disability score was 54.30 (SD 21.62). The following average pain intensity and disability scores after 12 months were similar to the values at6 months: 44.48 (SD 21.74) for pain intensity and 52.36 (SD 24.53) for disability. The Von Korff pain score at 6 months and after 1 year depends on gender and on the initial Von Korff pain score (p = 0.02). The Von Korff disability score at 6 months depends on gender, the baseline pain score and the baseline disability score (p = 0.001). Conclusions: our patients reported long-lasting pain that had a severe effect on their daily routines, and they could not return to their normal status prior to injury.

Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations.

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4(+) T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

Raman Spectroscopy, X-ray Diffraction, and Scanning Electron Microscopy as Noninvasive Methods for Microstructural Alterations in Psoriatic Nails.

Psoriasis is a chronic inflammatory disease associated with immune system dysfunction that can affect nails, with a negative impact on patient life quality. Usually, nail psoriasis is associated with skin psoriasis and is therefore relatively simple to diagnose. However, up to 10% of nail psoriasis occurs isolated and may be difficult to diagnose by means of current methods (nail biopsy, dermoscopy, video dermoscopy, capillaroscopy, ultrasound of the nails, etc.). Since the nail is a complex biological tissue, mainly composes of hard α-keratins, the structural and morphological techniques can be used to analyze the human fingernails. The aim of this study was to corroborate the information obtained using Raman spectroscopy with those obtained by scanning electron microscopy (SEM) and X-ray diffractometry and to assess the potential of these techniques as non-invasive dermatologic diagnostic tools and an alternative to current methods.

Genome-wide annotation of microRNA primary transcript structures reveals novel regulatory mechanisms.

Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through which miRNA expression is regulated has been greatly hindered by the incomplete annotation of primary miRNA (pri-miRNA) transcripts. While a subset of miRNAs are hosted in protein-coding genes, the majority of pri-miRNAs are transcribed as poorly characterized noncoding RNAs that are 10's to 100's of kilobases in length and low in abundance due to efficient processing by the endoribonuclease DROSHA, which initiates miRNA biogenesis. Accordingly, these transcripts are poorly represented in existing RNA-seq data sets and exhibit limited and inaccurate annotation in current transcriptome assemblies. To overcome these challenges, we developed an experimental and computational approach that allows genome-wide detection and mapping of pri-miRNA structures. Deep RNA-seq in cells expressing dominant-negative DROSHA resulted in much greater coverage of pri-miRNA transcripts compared with standard RNA-seq. A computational pipeline was developed that produces highly accurate pri-miRNA assemblies, as confirmed by extensive validation. This approach was applied to a panel of human and mouse cell lines, providing pri-miRNA transcript structures for 1291/1871 human and 888/1181 mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. These new assemblies uncovered unanticipated features and new potential regulatory mechanisms, including links between pri-miRNAs and distant protein-coding genes, alternative pri-miRNA splicing, and transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results dramatically expand our understanding of the organization of miRNA-encoding genes and provide a valuable resource for the study of mammalian miRNA regulation.

The genome of the blood fluke Schistosoma mansoni.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

DIAMUND: direct comparison of genomes to detect mutations.

DNA sequencing has become a powerful method to discover the genetic basis of disease. Standard, widely used protocols for analysis usually begin by comparing each individual to the human reference genome. When applied to a set of related individuals, this approach reveals millions of differences, most of which are shared among the individuals and unrelated to the disease being investigated. We have developed a novel algorithm for variant detection, one that compares DNA sequences directly to one another, without aligning them to the reference genome. When used to find de novo mutations in exome sequences from family trios, or to compare normal and diseased samples from the same individual, the new method, direct alignment for mutation discovery (DIAMUND), produces a dramatically smaller list of candidate mutations than previous methods, without losing sensitivity to detect the true cause of a genetic disease. We demonstrate our results on several example cases, including two family trios in which it correctly found the disease-causing variant while excluding thousands of harmless variants that standard methods had identified.

Xanthan-Polyurethane Conjugates: An Efficient Approach for Drug Delivery.

The antifungal agent, ketoconazole, and the anti-inflammatory drug, piroxicam, were incorporated into matrices of xanthan or oleic acid-esterified xanthan (Xn) and polyurethane (PU), to develop topical drug delivery systems. Compared to matrices without bioactive compounds, which only showed a nominal compressive stress of 32.18 kPa (sample xanthan-polyurethane) at a strain of 71.26%, the compressive resilience of the biomaterials increased to nearly 50.04 kPa (sample xanthan-polyurethane-ketoconazole) at a strain of 71.34%. The compressive strength decreased to around 30.67 kPa upon encapsulating a second drug within the xanthan-polyurethane framework (sample xanthan-polyurethane-piroxicam/ketoconazole), while the peak sustainable strain increased to 87.21%. The Weibull model provided the most suitable fit for the drug release kinetics. Unlike the materials based on xanthan-polyurethane, those made with oleic acid-esterified xanthan-polyurethane released the active ingredients more slowly (the release rate constant showed lower values). All the materials demonstrated antimicrobial effectiveness. Furthermore, a higher volume of piroxicam was released from oleic acid-esterified xanthan-polyurethane-piroxicam (64%) as compared to xanthan-polyurethane-piroxicam (44%). Considering these results, materials that include polyurethane and either modified or unmodified xanthan showed promise as topical drug delivery systems for releasing piroxicam and ketoconazole.

Detecting differential transcript usage in complex diseases with SPIT.

Differential transcript usage (DTU) plays a crucial role in determining how gene expression differs among cells, tissues, and developmental stages, contributing to the complexity and diversity of biological systems. In abnormal cells, it can also lead to deficiencies in protein function and underpin disease pathogenesis. Analyzing DTU via RNA sequencing (RNA-seq) data is vital, but the genetic heterogeneity in populations with complex diseases presents an intricate challenge due to diverse causal events and undetermined subtypes. Although the majority of common diseases in humans are categorized as complex, state-of-the-art DTU analysis methods often overlook this heterogeneity in their models. We therefore developed SPIT, a statistical tool that identifies predominant subgroups in transcript usage within a population along with their distinctive sets of DTU events. This study provides comprehensive assessments of SPIT's methodology and applies it to analyze brain samples from individuals with schizophrenia, revealing previously unreported DTU events in six candidate genes.

Combining DNA and protein alignments to improve genome annotation with LiftOn.

As the number and variety of assembled genomes continues to grow, the number of annotated genomes is falling behind, particularly for eukaryotes. DNA-based mapping tools help to address this challenge, but they are only able to transfer annotation between closely-related species. Here we introduce LiftOn, a homology-based software tool that integrates DNA and protein alignments to enhance the accuracy of genome-scale annotation and to allow mapping between relatively distant species. LiftOn's protein-centric algorithm considers both types of alignments, chooses optimal open reading frames, resolves overlapping gene loci, and finds additional gene copies where they exist. LiftOn can reliably transfer annotation between genomes representing members of the same species, as we demonstrate on human, mouse, honey bee, rice, and Arabidopsis thaliana. It can further map annotation effectively across species pairs as far apart as mouse and rat or Drosophila melanogaster and D. erecta.

Personalized Decisional Algorithms for Soft Tissue Defect Reconstruction after Abdominoperineal Resection for Low-Lying Rectal Cancers.

BACKGROUND: Abdominoperineal resection (APR)-the standard surgical procedure for low-lying rectal cancer (LRC)-leads to significant perineal defects, posing considerable reconstruction challenges that, in selected cases, necessitate the use of plastic surgery techniques (flaps). PURPOSE: To develop valuable decision algorithms for choosing the appropriate surgical plan for the reconstruction of perineal defects. METHODS: Our study included 245 LRC cases treated using APR. Guided by the few available publications in the field, we have designed several personalized decisional algorithms for managing perineal defects considering the following factors: preoperative radiotherapy, intraoperative position, surgical technique, perineal defect volume, and quality of tissues and perforators. The algorithms have been improved continuously during the entire period of our study based on the immediate and remote outcomes. RESULTS: In 239 patients following APR, the direct closing procedure was performed versus 6 cases in which we used various types of flaps for perineal reconstruction. Perineal incisional hernia occurred in 12 patients (5.02%) with direct perineal wound closure versus in none of those reconstructed using flaps. CONCLUSION: The reduced rate of postoperative complications suggests the efficiency of the proposed decisional algorithms; however, more extended studies are required to categorize them as evidence-based management guide tools.

A Histological Evaluation of the Efficiency of Using Periprosthetic Autologous Fat to Prevent Capsular Contracture Compared to Other Known Methods-An Experimental Study.

BACKGROUND: Capsular contracture remains a common complication in silicone breast implantation. The etiology, formation mechanisms, predisposing and favoring factors are still subjects of research. This study aims to demonstrate the effectiveness of using autologous fat introduced periprosthetically in preventing capsular contracture compared to other known methods: antibiotics and corticosteroids. METHODS: A cohort of 80 Wistar rats was included in the study, divided into four subgroups. All subjects received a silicone implant, implanted in a pocket created along the abdominal midline. The first subgroup served as the control group, with subjects having the implant placed without any treatment. For the second and third subgroups, the implants were treated with an antibiotic solution and intramuscular injections of dexamethasone, respectively. The subjects in the last subgroup received centrifuged autologous fat introduced periprosthetically. RESULTS: The subgroup with autologous fat exhibited a significantly smaller capsule thickness, which was poorly represented, with a smooth surface. The use of autologous fat for treating silicone prosthesis was linked with the lack of acute inflammation around the prosthetic site. CONCLUSIONS: Autologous fat helps to minimize the "non-self" reaction, which results in the development of a periprosthetic capsule consisting of mature collagen fibers interspersed with adipocytes.

Long-Term Clinical Outcomes of 3D-Printed Subperiosteal Titanium Implants: A 6-Year Follow-Up.

As an alternative to regenerative therapies, numerous authors have recently proposed bringing back subperiosteal implants. The aim of the study was to present our clinical experience with a subperiosteal jaw implant that needs minimal bone preparation and enables the rapid implantation of prosthetic teeth in edentulous, atrophic alveolar bone. The research included 36 complete or partial edentulous patients (61 subperiostal implants) over a period of 6 years. To create the patient-specific subperiostal implants design, DentalCAD 3.0 Galway software (exocad GmbH, Darmstadt, Germany) was used and fabricated with a Mysint 100 (Sisma S.p.A., Piovene Rocchette, Italy) by titanium alloy powder. The results showed that only 9 of the 36 cases were successful at 6-year follow-up, while 27 cases had complications, including exposure of the metal frame (early or delayed), mobility of the device prior to the first 4-6 months, and late mobility due to recurrent infections and progressive structure exposure; 1 case failed for reasons unrelated to the device. This study indicated that the prudent application of fully customized subperiosteal jaw implants is a dependable alternative for the dental rehabilitation of atrophic edentulous cases that necessitate bone grafts for traditional fixed dental implant solutions.