Mendel's terminology and notation reveal his understanding of genetics.

We describe both the terminology and use of symbols introduced by Mendel in his 1866 paper and discuss some misconceptions concerning their interpretation.

TNF-inhibitors or bisphosphonates in chronic nonbacterial osteomyelitis? - Results of an international retrospective multicenter study.

Chronic nonbacterial osteomyelitis (CNO) can cause significant morbidity, including bone pain and damage. In the absence of clinical trials, treatments include non-steroidal anti-inflammatory drugs, corticosteroids, TNF-inhibitors (TNFi) and/or bisphosphonates. In a retrospective chart review in the United Kingdom and Germany, we investigated response to TNFi and/or pamidronate. Ninety-one patients were included, receiving pamidronate (n = 47), TNFi (n = 22) or both sequentially (n = 22). Patients with fatigue [p = 0.003] and/or arthritis [p = 0.002] were more frequently treated with TNFi than pamidronate. Both therapies were associated with clinical remission at 6 months, and reduction of bone lesions on MRI at 12 months. While not reaching statistical significance, pamidronate resulted in faster resolution of MRI lesions. Fewer flares were observed with TNFi. Failure to respond to pamidronate was associated with female sex [p = 0.027], more lesions on MRI [p = 0.01] and higher CRP levels [p = 0.03]. Randomized clinical trials are needed to confirm observations and generate evidence.

COVID-19 vaccination intent among London healthcare workers.

BACKGROUND: The 10-month timeline from conception to regulatory approval of the Pfizer-BioNTech vaccine against SARS-CoV-2 is unprecedented in modern medicine. However, the climate of the pandemic has also seen anti-vaccination sentiments flourish. AIMS: To determine the intent to accept COVID-19 vaccination among healthcare workers at a London Hospital Trust and examine variation in uptake between demographic groups. METHODS: We conducted a cross-sectional survey open to staff working at the trust. Staff rated on a five-point scale the likelihood of them accepting COVID-19 vaccination. RESULTS: We received 514 responses, representing 16% of the workforce. About 59% of staff intended to seek vaccination, 24% to reject and 17% were unsure. There was significantly reduced intended uptake in females, younger age groups, healthcare assistants, nurses, staff of black ethnic backgrounds and those who rejected influenza vaccination. Safety was the dominant concern. CONCLUSIONS: Our study finds COVID-19 vaccinate hesitancy is prevalent among healthcare workers at a London Hospital Trust. It is particularly concerning that hesitancy was highest amongst groups most exposed to COVID-19 and most at risk of severe disease. Reasons behind disparities in uptake must be addressed to protect staff and prevent deepening inequalities within the healthcare workforce.

Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay.

PURPOSE: The prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification. METHODS: Freshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density. RESULTS: A nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used. CONCLUSIONS: Freshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.

Optimizing hepcidin measurement with a proficiency test framework and standardization improvement.

Objectives: Hepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization. Methods: We developed a pilot PT together with the Dutch EQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM. Results: The pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07±0.24 nmol/L. Conclusions: We established a framework for future PT to enable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.

Origin of Groundwater Arsenic in a Rural Pleistocene Aquifer in Bangladesh Depressurized by Distal Municipal Pumping.

Across South Asia, millions of villagers have reduced their exposure to high-arsenic (As) groundwater by switching to low-As wells. Isotopic tracers and flow modeling are used in this study to understand the groundwater flow system of a semi-confined aquifer of Pleistocene (>10 kyr) age in Bangladesh that is generally low in As but has been perturbed by massive pumping at a distance of about 25 km for the municipal water supply of Dhaka. A 10- to 15-m-thick clay aquitard caps much of the intermediate aquifer (>40- to 90-m depth) in the 3-km2 study area, with some interruptions by younger channel sand deposits indicative of river scouring. Hydraulic heads in the intermediate aquifer below the clay-capped areas are 1-2 m lower than in the high-As shallow aquifer above the clay layer. In contrast, similar heads in the shallow and intermediate aquifer are observed where the clay layer is missing. The head distribution suggests a pattern of downward flow through interruptions in the aquitard and lateral advection from the sandy areas to the confined portion of the aquifer. The interpreted flow system is consistent with 3H-3He ages, stable isotope data, and groundwater flow modeling. Lateral flow could explain an association of elevated As with high methane concentrations within layers of gray sand below certain clay-capped portions of the Pleistocene aquifer. An influx of dissolved organic carbon from the clay layer itself leading to a reduction of initially orange sands has also likely contributed to the rise of As.

Mendel's pea crosses: varieties, traits and statistics.

A controversy arose over Mendel's pea crossing experiments after the statistician R.A. Fisher proposed how these may have been performed and criticised Mendel's interpretation of his data. Here we re-examine Mendel's experiments and investigate Fisher's statistical criticisms of bias. We describe pea varieties available in Mendel's time and show that these could readily provide all the material Mendel needed for his experiments; the characters he chose to follow were clearly described in catalogues at the time. The combination of character states available in these varieties, together with Eichling's report of crosses Mendel performed, suggest that two of his F3 progeny test experiments may have involved the same F2 population, and therefore that these data should not be treated as independent variables in statistical analysis of Mendel's data. A comprehensive re-examination of Mendel's segregation ratios does not support previous suggestions that they differ remarkably from expectation. The χ2 values for his segregation ratios sum to a value close to the expectation and there is no deficiency of extreme segregation ratios. Overall the χ values for Mendel's segregation ratios deviate slightly from the standard normal distribution; this is probably because of the variance associated with phenotypic rather than genotypic ratios and because Mendel excluded some data sets with small numbers of progeny, where he noted the ratios "deviate not insignificantly" from expectation.

Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum).

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available.

The Impact of Aquifer Flushing on Groundwater Arsenic Across a 35-km Transect Perpendicular to the Upper Brahmaputra River in Assam, India.

Well testing in the floodplain of the Brahmaputra River in Golaghat and Jorhat districts of Assam, India, shows that groundwater arsenic (As) concentrations increase with distance from the river. To establish the origin of this pattern, an additional 900 wells <60 m deep were tested for As and 9 sites were drilled along a 35-km transect perpendicular to the river. The field data show no relation between groundwater As concentrations ranging from <1 to 660 µg/L along the transect and (a) As concentrations of <1-5 mg/kg in cuttings of aquifer sand recovered while drilling or (b) the degree of reduction of iron oxides in these cuttings. The drilling indicates, however, a marked increase in the thickness of a clay layer capping the aquifer starting from <1-5 m near the river to over 60 m at the most distant site towards the base of the Naga foothills. Organic radiocarbon ages of 18-46 kyr obtained from all but one of 13 clay samples indicate pre-Holocene deposition of the underlying sands across the entire transect. Radiocarbon ages of dissolved inorganic carbon of 0.2, 4.7, and 17.8 kyr were measured in groundwater from 3 monitoring wells installed to 30-60 m depth at distances of 10, 20, and 40 km from the river, respectively. A conceptual groundwater flow model consistent with monitored heads and groundwater ages suggests that thick clay layers capping the aquifer further from the river inhibited flushing of the aquifer and, as a result, preserved higher As levels in groundwater.

Renal Transplantation With Final Allocation Based on the Virtual Crossmatch.

Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However, the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor-recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long-term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor-specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within 1 year (AR <1 year) for FCXM+ (n = 54) and FCXM- (n = 454) recipients. Median follow-up is 7.1 years. FCXM+ recipients were significantly different from FCXM- recipients for the following risk factors: living donor (24% vs. 39%, p = 0.03), duration of dialysis (31.0 months vs. 13.5 months, p = 0.008), retransplants (17% vs. 7.3%, p = 0.04), % sensitized (63% vs. 19%, p = 0.001), and PRA >80% (20% vs. 4.8%, p = 0.001). Despite these differences, 5-year actual graft survival rates are 87% and 84%, respectively. AR <1 year occurred in 13% FCXM+ and 12% FCXM- recipients. Crossmatch status was not associated with graft outcomes in any univariate or multivariate model. Renal transplantation can be performed successfully, using SPI as the definitive test for donor-recipient compatibility.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.

Regulatory T cell (Treg)-based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3(+) Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.

NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes.

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

Diagnosis and management of antibody-mediated rejection: current status and novel approaches.

Advances in multimodal immunotherapy have significantly reduced acute rejection rates and substantially improved 1-year graft survival following renal transplantation. However, long-term (10-year) survival rates have stagnated over the past decade. Recent studies indicate that antibody-mediated rejection (ABMR) is among the most important barriers to improving long-term outcomes. Improved understanding of the roles of acute and chronic ABMR has evolved in recent years following major progress in the technical ability to detect and quantify recipient anti-HLA antibody production. Additionally, new knowledge of the immunobiology of B cells and plasma cells that pertains to allograft rejection and tolerance has emerged. Still, questions regarding the classification of ABMR, the precision of diagnostic approaches, and the efficacy of various strategies for managing affected patients abound. This review article provides an overview of current thinking and research surrounding the pathophysiology and diagnosis of ABMR, ABMR-related outcomes, ABMR prevention and treatment, as well as possible future directions in treatment.

Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells.

Manipulating gene expression in mammalian cells is critical for cell engineering applications. Here we explore the potential of transcription factor (TF) recognition element arrays as DNA tools for modifying free TF levels in cells and thereby controlling gene expression. We first demonstrate proof-of-concept, showing that Tet TF-binding recognition element (RE) arrays of different lengths can tune gene expression and alter gene circuit performance in a predictable manner. We then open-up the approach to interface with any TF with a known binding site by developing a new method called Cloning Troublesome Repeats in Loops (CTRL) that can assemble plasmids with up to 256 repeats of any RE sequence. Transfection of RE array plasmids assembled by CTRL into mammalian cells show potential to modify host cell gene regulation at longer array sizes by sequestration of the TF of interest. RE array plasmids built using CTRL were demonstrated to target both synthetic and native mammalian TFs, illustrating the ability to use these tools to modulate genetic circuits and instruct cell fate. Together this work advances our ability to assemble repetitive DNA arrays and showcases the use of TF-binding RE arrays as a method for manipulating mammalian gene expression, thus expanding the possibilities for mammalian cell engineering.

Experiences from the implementation of physical therapy via telehealth for individuals with Parkinson disease during the COVID-19 pandemic.

PURPOSE: To (1) determine the characteristics and participation rate of adults with Parkinson disease (PD) in physical therapy (PT) delivered via telehealth, (2) identify the outcome measures and interventions implemented, (3) determine the safety of and (4) patient and therapist satisfaction with PT via telehealth in a clinic specializing in the care of people with PD during the coronavirus pandemic. MATERIALS & METHODS: A retrospective analysis of PT services via telehealth was conducted. Participating patients completed a satisfaction survey. Physical therapists (PTs) who delivered this care were interviewed. Three coders conducted thematic analysis of interviews. Descriptive statistics described the participation rate, demographics, outcome measures, interventions, and safety. RESULTS: There was a 71.4% participation rate. Participants (n = 55) were white (96%), non-Hispanic (100%), older adult (mean = 69.5 years (8.3)) males (65.5%). Non-participants (n = 22) had similar demographics. Therapists selected patient-reported measures more often than performance-based measures. Therapeutic exercise was the most common intervention. All patients (80% response rate) reported satisfaction with their experience. PTs reported the home enhanced specificity of training but impeded evaluation. Therapists endorsed a hybrid model for future practice. CONCLUSIONS: Patients reported satisfaction with PT via telehealth during the pandemic. A hybrid model may support optimal delivery of PT.IMPLICATIONS FOR REHABILITATIONPhysical therapy via telehealth for patients with Parkinson disease was acceptable to patients and physical therapists in our study.Physical therapy via telehealth was safe for people with Parkinson disease in our study, although availability and benefits may not be reaching all populations equitably.Both physical therapists and patients endorse a hybrid model of care (a combination of in-person and remote assessment and treatment) to profit from the strengths of in-person and virtual formats while minimizing barriers to access.

Dahlia latent viroid: a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification.

A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genus Pospiviroid, family Pospiviroidae, reported in this ornamental previously. RT-PCR with primers designed for amplifying all pospiviroids produced no amplicons, but RT-PCR with random primers revealed a 342 nt RNA. The sequence of this RNA was confirmed with specific primers, which additionally revealed its presence in many dahlia cultivars. The RNA was named Dahlia latent viroid (DLVd) because it replicates autonomously, but symptomlessly, in dahlia and shares maximum sequence identity with other viroids of less than 56 %. Furthermore, DLVd displays characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR), and the ability to form the metastable structures hairpins I and II. Its CCR is identical to that of Hop stunt viroid (HSVd, genus Hostuviroid). However, DLVd: (i) has the terminal conserved region present in members of the genus Pospiviroid, but absent in HSVd, and (ii) lacks the terminal conserved hairpin present in HSVd. Phylogenetic reconstructions indicate that HSVd and Pepper chat fruit viroid (genus Pospiviroid) are the closest relatives of DLVd, but DLVd differs from these viroids in its host range, restricted to dahlia so far. Therefore, while DLVd fulfils the criteria to be a novel species of the family Pospiviroidae, its recombinant origin makes assignment to the genera Pospiviroid or Hostuviroid problematic.

The B gene of pea encodes a defective flavonoid 3',5'-hydroxylase, and confers pink flower color.

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

Evaluation of the vaccination efficacy against H5N1 in domestic poultry in the Red River Delta in Vietnam.

The domestic poultry population in Vietnam has been vaccinated against highly pathogenic avian influenza (HPAI) H5N1 since 2005. Since then, outbreaks have continued to occur without a clear understanding of the mechanisms involved. The general objective of this study was to understand the epidemiology of the disease in the context of vaccination and to draw some conclusions about vaccination efficacy in the domestic poultry population of the Red River Delta area. Five cross-sectional surveys to measure the serological and virological prevalence in vaccinated and unvaccinated poultry were performed from the end of 2008 to June 2010. The global seroprevalence was 24% (95% confidence interval 19·9-28·2). Determinants of vaccine immunogenicity were identified separately in chickens and ducks as well as determinants of the seroconversion in unvaccinated birds. The results highlight the difficulties in maintaining good flock immunity in poultry populations using inactivated vaccine in the field with two vaccination rounds per year, and in preventing circulation of virus in co-existing unvaccinated poultry.

Efficacy of surgery for internal snapping hip.

Our purpose was to evaluate evidence regarding the effectiveness of open iliopsoas release and lengthening with arthroscopic approaches, which combine iliopsoas release with the treatment of intra-articular pathology as treatments for internal snapping hip syndrome. Searches were performed of 4 databases and 12 reports on the surgical treatment of internal snapping hip were reviewed. The authors tabulated data according to year of study, journal, study type, level of evidence, patient demographics, procedure, and outcomes. We found that surgical treatment of internal snapping hip improves patient symptoms in the majority of patients. Endoscopic release is associated with fewer reported complications compared with open release. All studies of endoscopic treatment of internal snapping hip syndrome also performed intra-articular examination and treatment of intra-articular pathology. The incidence of associated intra-articular pathology was reported in 5 of 6 studies. These results suggest that a painful snapping iliopsoas tendon in many cases may be a result of intra-articular hip pathology rather than an independent entity. Early studies suggest no difference in efficacy or associated complications rate when comparing extracapsular to transcapsular iliopsoas tendon release. Further studies are needed comparing operative to nonoperative treatment and methods of determining patients likely to benefit from operative intervention.