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Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples-a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.

Xia-Gibbs syndrome (XGS; MIM# 615829) is a rare mendelian disorder characterized by Development Delay (DD), intellectual disability (ID), and hypotonia. Individuals with XGS typically harbor de novo protein-truncating mutations in the AT-Hook DNA binding motif containing 1 (AHDC1) gene, although some missense mutations can also cause XGS. Large de novo heterozygous deletions that encompass the AHDC1 gene have also been ascribed as diagnostic for the disorder, without substantial evidence to support their pathogenicity. We analyzed 19 individuals with large contiguous deletions involving AHDC1, along with other genes. One individual bore the smallest known contiguous AHDC1 deletion (∼350 Kb), encompassing eight other genes within chr1p36.11 (Feline Gardner-Rasheed, IFI6, FAM76A, STX12, PPP1R8, THEMIS2, RPA2, SMPDL3B) and terminating within the first intron of AHDC1. The breakpoint junctions and phase of the deletion were identified using both short and long read sequencing (Oxford Nanopore). Quantification of RNA expression patterns in whole blood revealed that AHDC1 exhibited a mono-allelic expression pattern with no deficiency in overall AHDC1 expression levels, in contrast to the other deleted genes, which exhibited a 50% reduction in mRNA expression. These results suggest that AHDC1 expression in this individual is compensated by a novel regulatory mechanism and advances understanding of mutational and regulatory mechanisms in neurodevelopmental disorders., (© 2022 Wiley Periodicals LLC.)

Background: Cryptosporidium parvum is an apicomplexan parasite commonly found across many host species with a global infection prevalence in human populations of 7.6%. Understanding its diversity and genomic makeup can help in fighting established infections and prohibiting further transmission. The basis of every genomic study is a high-quality reference genome that has continuity and completeness, thus enabling comprehensive comparative studies., Findings: Here, we provide a highly accurate and complete reference genome of Cryptosporidium parvum. The assembly is based on Oxford Nanopore reads and was improved using Illumina reads for error correction. We also outline how to evaluate and choose from different assembly methods based on 2 main approaches that can be applied to other Cryptosporidium species. The assembly encompasses 8 chromosomes and includes 13 telomeres that were resolved. Overall, the assembly shows a high completion rate with 98.4% single-copy BUSCO genes., Conclusions: This high-quality reference genome of a zoonotic IIaA17G2R1 C. parvum subtype isolate provides the basis for subsequent comparative genomic studies across the Cryptosporidium clade. This will enable improved understanding of diversity, functional, and association studies., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Cryo-electron microscopy (cryo-EM) is rapidly becoming an attractive method in the field of structural biology. With the exploding popularity of cryo-EM, sample preparation must evolve to prevent congestion in the workflow. The dire need for improved microscopy samples has led to a diversification of methods. This Review aims to categorize and explain the principles behind various techniques in the preparation of vitrified samples for the electron microscope. Various aspects and challenges in the workflow are discussed, from sample optimization and carriers to deposition and vitrification. Reliable and versatile specimen preparation remains a challenge, and we hope to give guidelines and posit future directions for improvement.

Xia-Gibbs syndrome (XGS) is a rare Mendelian disease typically caused by de novo stop-gain or frameshift mutations in the AT-hook DNA binding motif containing 1 (AHDC1) gene. Patients usually present in early infancy with hypotonia and developmental delay and later exhibit intellectual disability (ID). The overall presentation is variable, however, and the emerging clinical picture is still evolving. A detailed phenotypic analysis of 34 XGS individuals revealed five core phenotypes (delayed motor milestones, speech delay, low muscle tone, ID, and hypotonia) in more than 80% of individuals and an additional 12 features that occurred more variably. Seizures and scoliosis were more frequently associated with truncations that arise before the midpoint of the protein although the occurrence of most features could not be predicted by the mutation position. Transient expression of wild type and different patient truncated AHDC1 protein forms in human cell lines revealed abnormal patterns of nuclear localization including a diffuse distribution of a short truncated form and nucleolar aggregation in mid-protein truncated forms. Overall, both the occurrence of variable phenotypes and the different distribution of the expressed protein reflect the heterogeneity of this syndrome., (© 2021 Wiley Periodicals LLC.)

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

Workplace violence and aggression are receiving increasing attention, especially when perpetrated in at-risk services such as psychiatric and emergency departments. Many healthcare providers have been victims of verbal aggressions (VAs) and physical aggressions (PAs), as well as injuries (INs), at the hands of patients. We conducted a 1-year retrospective questionnaire-based survey to assess workplace violence and aggression experienced by staff working at the Psychiatric Service of the Health District of Bolzano-Bozen (Italy). We performed parametric statistics. Logistic regression estimated the size of the association between PA occurrence and staff characteristics. Our psychiatric service's employees were frequent victims (91.5%) of 1 or more aggression/injury in the previous year. VAs and INs showed comparable frequencies among the three sites of our service, that is, the inpatient (INP), the outpatient (OUTP), and the rehabilitation (REHAB) units, differently from PAs, which were more common at INP (p < 0.001). The logistic regression model showed that female sex, working at INP, and a shorter psychiatry work experience predicted PAs occurrence. Most of the staff felt they could benefit from aggression management. Findings show that VAs, PAs, and INs are common among our psychiatric service's employees and point to the need to provide staff training on de-escalation.

Tissue decellularisation has gained much attention in regenerative medicine as an alternative to synthetic materials. In decellularised tissues, biological cues can be maintained and provide cellular environments still unmet by synthetic materials. Supercritical CO 2 (scCO 2 ) has recently emerged as a promising alternative decellularisation technique to aggressive detergents; in addition, scCO 2 provides innate sterilisation. However, to date, decellularisation with scCO 2 is limited to only a few tissue types with low cellular density. In the current study, a scCO 2 technique to decellularise high density tissues, including articular cartilage, tendon and skin, was developed. Results showed that most of the cellular material was removed, while the sample structure and biocompatibility was preserved. The DNA content was reduced in cartilage, tendon and skin as compared to the native tissue. The treatment did not affect the initial tendon elastic modulus [reduced from 126.35 ± 9.79 MPa to 113.48 ± 8.48 MPa (p 〉 0.05)], while it reduced the cartilage one [from 12.06 ± 2.14 MPa to 1.17 ± 0.34 MPa (p 〈 0.0001)]. Interestingly, cell adhesion molecules such as fibronectin and laminin were still present in the tissues after decellularisation. Bovine chondrocytes were metabolically active and adhered to the surface of all decellularised tissues after 1 week of cell culture. The developed method has the potential to become a cost-effective, one-step procedure for the decellularisation of dense tissues.

Leptospirosis is a neglected zoonotic disease with worldwide endemicity and continues to be a significant public health burden on resource-limited populations. Previously, we produced three highly purified recombinant antigens (rLipL32, rLipL41, and rLigA-Rep) and evaluated their performance of detecting Leptospira -specific antibodies in enzyme-linked immunosorbent assay (ELISA) as compared with the microscopic agglutination test (MAT). The overall sensitivity of this assay approached 90%. Recently, another recombinant antigen (rLigB-Rep) was prepared. We tested each individual antigen and a 1:1:1:1 mixture of these four antigens for the detection of Leptospira -specific antibodies in ELISA. The performance of these recombinant antigens was evaluated with a much larger febrile patient panel (337 MAT-confirmed positive sera and 92 MAT-negative sera from febrile patients). Combining the detection results of immunoglobulin M and immunoglobulin G from these four individual antigens, the overall sensitivity was close to 90% but the specificity was only 66%, based on the MAT reference method. The overall sensitivity and specificity of the four-antigen mixture were 82% and 86%, respectively. The mixture of four antigens also exhibited a broader reactivity with MAT-positive samples of 18 serovars from six major pathogenic Leptospira species. Given the limitations of MAT, the data were further analyzed by Bayesian latent class model, showing that ELISA using a 1:1:1:1 mixture still maintained high sensitivity (79%) and specificity (88%) as compared with the sensitivity (90%) and specificity (83%) of MAT. Therefore, ELISA using a mixture of these four antigens could be a very useful test for seroprevalence studies.

Contractile stress generation by adherent cells is largely determined by the interplay of forces within their cytoskeleton. It is known that actin stress fibers, connected to focal adhesions, provide contractile stress generation, while microtubules and intermediate filaments provide cells compressive stiffness. Recent studies have shown the importance of the interplay between the stress fibers and the intermediate filament vimentin. Therefore, the effect of the interplay between the stress fibers and vimentin on stress generation was quantified in this study. We hypothesized that net stress generation comprises the stress fiber contraction combined with the vimentin resistance. We expected an increased net stress in vimentin knockout (VimKO) mouse embryonic fibroblasts (MEFs) compared to their wild-type (vimentin wild-type (VimWT)) counterparts, due to the decreased resistance against stress fiber contractility. To test this, the net stress generation by VimKO and VimWT MEFs was determined using the thin film method combined with sample-specific finite element modeling. Additionally, focal adhesion and stress fiber organization were examined via immunofluorescent staining. Net stress generation of VimKO MEFs was three-fold higher compared to VimWT MEFs. No differences in focal adhesion size or stress fiber organization and orientation were found between the two cell types. This suggests that the increased net stress generation in VimKO MEFs was caused by the absence of the resistance that vimentin provides against stress fiber contraction. Taken together, these data suggest that vimentin resists the stress fiber contractility, as hypothesized, thus indicating the importance of vimentin in regulating cellular stress generation by adherent cells.

The accumulation of sequenced Francisella strains has made it increasingly apparent that the 16S rRNA gene alone is not enough to stratify the Francisella genus into precise and clinically useful classifications. Continued whole-genome sequencing of isolates will provide a larger base of knowledge for targeted approaches with broad applicability. Additionally, examination of genomic information on a case-by-case basis will help resolve outstanding questions regarding strain stratification. We report the complete genome sequence of a clinical isolate, designated here as F. novicida-like strain TCH2015, acquired from the lymph node of a 6-year-old male. Two features were atypical for F. novicida: exhibition of functional oxidase activity and additional gene content, including proposed virulence determinants. These differences, which could potentially impact virulence and clinical diagnosis, emphasize the need for more comprehensive methods to profile Francisella isolates. This study highlights the value of whole-genome sequencing, which will lead to a more robust database of environmental and clinical genomes and inform strategies to improve detection and classification of Francisella strains., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Scrub typhus (ST) is an infection caused by Orientia tsutsugamushi. Historically, ST was ranked as the second most important arthropod-borne medical problem only behind malaria during World War II and the Vietnam War. The disease occurs mainly in Southeast Asia and has been shown to emerge and reemerge in new areas, implying the increased risk for U.S. military and civilian personnel deployed to these regions. ST can effectively be treated by doxycycline provided the diagnosis is made early, before the development of severe complications. Scrub Typhus Detect is a lateral flow rapid test based on a mixture of recombinant 56-kDa antigens with broad reactivity. The performance of this prototype product was evaluated against indirect immunofluorescence assay, the serological gold standard. Using 249 prospectively collected samples from Thailand, the sensitivity and specificity for IgM was found to be 100% and 92%, respectively, suggesting a high potential of this product for clinical use. This product will provide a user friendly, rapid, and accurate diagnosis of ST for clinicians to provide timely and accurate treatments of deployed personnel., (Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.)

Leptospirosis is considered to be the most widespread zoonosis. This worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus Leptospira. Polymerase chain reaction (PCR)-based diagnostic assays have been developed for detecting Leptospira DNA in cell cultures and clinical samples. Because PCR requires specialized equipment and extensive end-user training, it is not suitable for routine work in resource-limited areas. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of Leptospira in patient, animal and environmental samples using lyophilized reagents at a single temperature of around 63°C with a heating block. The sensitivity of this LAMP assay is very similar to the PCR method. The amplified DNA products can be visualized with the naked eyes using hydroxy naphthol blue or detected by the fluorescence signal of SYBR green dye in the reaction when an ultraviolet lamp or compact fluorescence tube scanner is used. This LAMP assay is simple, rapid, and can be performed with a water bath or heating block. The lyophilized LAMP reagents are stable for 3 months when stored at 4°C and 1 month when stored at 25°C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic., (Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.)

The bacterial pathogen Francisella tularensis was recently renewed as a tier-one select agent. F. tularensis subsp. tularensis (type A) and holarctica (type B) are of clinical relevance. Here, we report the complete genome of a virulent F. tularensis type B strain and describe its usefulness in comparative genomics., (Copyright © 2015 Atkins et al.)

Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

Several lines of evidence support the presence of dosage-sensitive genes on chromosome 21 that regulate leukemogenesis and hematopoiesis. We report a detailed clinical and molecular characterization of 3 patients with chronic thrombocytopenia caused by distinct constitutional microdeletions involving chromosomal region 21q22.12. The patients exhibited growth restriction, dysmorphic features, and developmental delays. One patient developed acute myelogenous leukemia (AML) at 6 years of age. All 3 deletions included the RUNX1, CLIC6, DSCR, and KCNE1 genes. Our data provide additional support for the role of RUNX1 haploinsufficiency in megakaryopoiesis and predisposition to AML. The leukemic clone had trisomy 21 resulting from duplication of chromosome 21 containing the RUNX1 deletion. This shows that genes other than RUNX1 must also play a role in AML associated with trisomy 21. We recommend that children with syndromic thrombocytopenia have clinical array-comparative genomic hybridization analysis and appropriate cytogenetic studies to facilitate our ability to provide a definitive diagnosis.

The duplication 17p11.2 syndrome, associated with dup(17)(p11.2p11.2), is a recently recognized syndrome of multiple congenital anomalies and mental retardation and is the first predicted reciprocal microduplication syndrome described--the homologous recombination reciprocal of the Smith-Magenis syndrome (SMS) microdeletion (del(17)(p11.2p11.2)). We previously described seven subjects with dup(17)(p11.2p11.2) and noted their relatively mild phenotype compared with that of individuals with SMS. Here, we molecularly analyzed 28 additional patients, using multiple independent assays, and also report the phenotypic characteristics obtained from extensive multidisciplinary clinical study of a subset of these patients. Whereas the majority of subjects (22 of 35) harbor the homologous recombination reciprocal product of the common SMS microdeletion (~3.7 Mb), 13 subjects (~37%) have nonrecurrent duplications ranging in size from 1.3 to 15.2 Mb. Molecular studies suggest potential mechanistic differences between nonrecurrent duplications and nonrecurrent genomic deletions. Clinical features observed in patients with the common dup(17)(p11.2p11.2) are distinct from those seen with SMS and include infantile hypotonia, failure to thrive, mental retardation, autistic features, sleep apnea, and structural cardiovascular anomalies. We narrow the critical region to a 1.3-Mb genomic interval that contains the dosage-sensitive RAI1 gene. Our results refine the critical region for Potocki-Lupski syndrome, provide information to assist in clinical diagnosis and management, and lend further support for the concept that genomic architecture incites genomic instability.