CT image-based biomarkers for opportunistic screening of osteoporotic fractures: a systematic review and meta-analysis.

The use of opportunistic computed tomography (CT) image-based biomarkers may be a low-cost strategy for screening older individuals at high risk for osteoporotic fractures and populations that are not sufficiently targeted. This review aimed to assess the discriminative ability of image-based biomarkers derived from existing clinical routine CT scans for hip, vertebral, and major osteoporotic fracture prediction. A systematic search in PubMed MEDLINE, Embase, Cochrane, and Web of Science was conducted from the earliest indexing date until July 2023. The evaluation of study quality was carried out using a modified Quality Assessment Tool for Diagnostic Accuracy Studies (QUADAS-2) checklist. The primary outcome of interest was the area under the curve (AUC) and its corresponding 95% confidence intervals (CIs) obtained for four main categories of biomarkers: areal bone mineral density (BMD), image attenuation, volumetric BMD, and finite element (FE)-derived biomarkers. The meta-analyses were performed using random effects models. Sixty-one studies were included in this review, among which 35 were synthesized in a meta-analysis and the remaining articles were qualitatively synthesized. In comparison to the pooled AUC of areal BMD (0.73 [95% CI 0.71-0.75]), the pooled AUC values for predicting osteoporotic fractures for FE-derived parameters (0.77 [95% CI 0.72-0.81]; p < 0.01) and volumetric BMD (0.76 [95% CI 0.71-0.81]; p < 0.01) were significantly higher, but there was no significant difference with the pooled AUC for image attenuation (0.73 [95% CI 0.66-0.79]; p = 0.93). Compared to areal BMD, volumetric BMD and FE-derived parameters may provide a significant improvement in the discrimination of osteoporotic fractures using opportunistic CT assessments.

Refracture and mortality risk in the elderly with osteoporotic fractures: the AGES-Reykjavik study.

There is imminent refracture risk in elderly individuals for up to six years, with a decline thereafter except in women below 75 who face a constant elevated risk. Elderly men with fractures face the highest mortality risk, particularly those with hip and vertebral fractures. Targeted monitoring and treatment strategies are recommended. PURPOSE: Current management and interventions for osteoporotic fractures typically focus on bone mineral density loss, resulting in suboptimal evaluation of fracture risk. The aim of the study is to understand the progression of fractures to refractures and mortality in the elderly using multi-state models to better target those at risk. METHODS: This prospective, observational study analysed data from the AGES-Reykjavik cohort of Icelandic elderly, using multi-state models to analyse the evolution of fractures into refractures and mortality, and to estimate the probability of future events in subjects based on prognostic factors. RESULTS: At baseline, 4778 older individuals aged 65 years and older were included. Elderly men, and elderly women above 80 years of age, had a distinct imminent refracture risk that lasted between 2-6 years, followed by a sharp decline. However, elderly women below 75 continued to maintain a nearly constant refracture risk profile for ten years. Hip (30-63%) and vertebral (24-55%) fractures carried the highest 5-year mortality burden for elderly men and women, regardless of age, and for elderly men over 80, lower leg fractures also posed a significant mortality risk. CONCLUSION: The risk of refracture significantly increases in the first six years following the initial fracture. Elderly women, who experience fractures at a younger age, should be closely monitored to address their long-term elevated refracture risk. Elderly men, especially those with hip and vertebral fractures, face substantial mortality risk and require prioritized monitoring and treatment.

Next Generation Sequencing of the Pig αß TCR Repertoire Identifies the Porcine Invariant NKT Cell Receptor.

Swine represent the only livestock with an established invariant NKT (iNKT) cell-CD1d system. In this study, we exploited the fact that pig iNKT cells can be purified using a mouse CD1d tetramer reagent to establish their TCR repertoire by next generation sequencing. CD1d tetramer-positive pig cells predominantly expressed an invariant Vα-Jα rearrangement, without nontemplate nucleotide diversity, homologous to the Vα24-Jα18 and Vα14-Jα18 rearrangements of human and murine iNKT cells. The coexpressed ß-chain used a Vß segment homologous to the semivariant Vß11 and Vß8.2 segments of human and murine iNKT cell receptors. Molecular modeling found that contacts within CD1d and CDR1α that underlie fine specificity differences between mouse and human iNKT cells are conserved between pigs and humans, indicating that the response of porcine and human iNKT cells to CD1d-restricted Ags may be similar. Accordingly, pigs, which are an important species for diverse fields of biomedical research, may be useful for developing human-based iNKT cell therapies for cancer, infectious diseases, and other disorders. Our study also sequenced the expressed TCR repertoire of conventional porcine αß T cells, which identified 48 Vα, 50 Jα, 18 Vß, and 18 Jß sequences, most of which correspond to human gene segments. These findings provide information on the αß TCR usage of pigs, which is understudied and deserves further attention.

YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.

In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.

Effect of Statistically Iterative Image Reconstruction on Vertebral Bone Strength Prediction Using Bone Mineral Density and Finite Element Modeling: A Preliminary Study.

Statistical iterative reconstruction (SIR) using multidetector computed tomography (MDCT) is a promising alternative to standard filtered back projection (FBP), because of lower noise generation while maintaining image quality. Hence, we investigated the feasibility of SIR in predicting MDCT-based bone mineral density (BMD) and vertebral bone strength from finite element (FE) analysis. The BMD and FE-predicted bone strength derived from MDCT images reconstructed using standard FBP (FFBP) and SIR with (FSIR) and without regularization (FSIRB0) were validated against experimental failure loads (Fexp). Statistical iterative reconstruction produced the best quality images with regard to noise, signal-to-noise ratio, and contrast-to-noise ratio. Fexp significantly correlated with FFBP, FSIR, and FSIRB0. FFBP had a significant correlation with FSIRB0 and FSIR. The BMD derived from FBP, SIRB0, and SIR were significantly correlated. Effects of regularization should be further investigated with FE and BMD analysis to allow for an optimal iterative reconstruction algorithm to be implemented in an in vivo scenario.

Analysis of Mitochondrial DNA Polymorphisms in the Human Cell Lines HepaRG and SJCRH30.

The mitochondrial DNA (mtDNA) sequences of two commonly used human cell lines, HepaRG and SJCRH30, were determined. HepaRG originates from a liver tumor obtained from a patient with hepatocarcinoma and hepatitis C while SJCRH30 originates from a rhabdomyosarcoma patient tumor. In comparison to the revised Cambridge Reference Sequence, HepaRG and SJCRH30 mtDNA each contain 14 nucleotide variations. In addition to an insertion of a cytosine at position 315 (315insC), the mtDNA sequences from both cell types share six common polymorphisms. Heteroplasmic variants were identified in both cell types and included the identification of the 315insC mtDNA variant at 42 and 75% heteroplasmy in HepaRG and SJCRH30, respectively. Additionally, a novel heteroplasmic G13633A substitution in the HepaRG ND5 gene was detected at 33%. Previously reported cancer-associated mtDNA variants T195C and T16519C were identified in SJCRH30, both at homoplasmy (100%), while HepaRG mtDNA harbors a known prostate cancer-associated T6253C substitution at near homoplasmy, 95%. Based on our sequencing analysis, HepaRG mtDNA is predicted to lie within haplogroup branch H15a1 while SJCRH30 mtDNA is predicted to localize to H27c. The catalog of polymorphisms and heteroplasmy reported here should prove useful for future investigations of mtDNA maintenance in HepaRG and SJCRH30 cell lines.

Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA.

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (<1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA. To understand the nature of heteroplasmy, we developed Mseek, a novel technique to purify and sequence mtDNA. Mseek yields high purity (>90%) mtDNA and its ability to detect rare variants is limited only by sequencing depth, providing unprecedented sensitivity and specificity. Using Mseek, we confirmed the ubiquity of heteroplasmy by analyzing mtDNA from a diverse set of cell lines and human samples. Applying Mseek to colonies derived from single cells, we find heteroplasmy is stably maintained in individual daughter cells over multiple cell divisions. We hypothesized that the stability of heteroplasmy could be facilitated by intercellular exchange of mtDNA. We explicitly demonstrate this exchange by co-culturing cell lines with distinct mtDNA haplotypes. Our results shed new light on the maintenance of heteroplasmy and provide a novel platform to investigate features of heteroplasmy in normal and diseased states.

High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries.

We introduce two large-scale resources for functional analysis of microRNA (miRNA): a decoy library for inhibiting miRNA function and a sensor library for monitoring microRNA activity. To take advantage of the sensor library, we developed a high-throughput assay called Sensor-seq to simultaneously quantify the activity of hundreds of miRNAs. Using this approach, we show that only the most abundant miRNAs in a cell mediate target suppression. Over 60% of detected miRNAs had no discernible activity, which indicated that the functional 'miRNome' of a cell is considerably smaller than currently inferred from profiling studies. Moreover, some highly expressed miRNAs exhibited relatively weak activity, which in some cases correlated with a high target-to-miRNA ratio or increased nuclear localization of the miRNA. Finally, we show that the miRNA decoy library can be used for pooled loss-of-function studies. These tools are valuable resources for studying miRNA biology and for miRNA-based therapeutics.

MiST: a new approach to variant detection in deep sequencing datasets.

MiST is a novel approach to variant calling from deep sequencing data, using the inverted mapping approach developed for Geoseq. Reads that can map to a targeted exonic region are identified using exact matches to tiles from the region. The reads are then aligned to the targets to discover variants. MiST carefully handles paralogous reads that map ambiguously to the genome and clonal reads arising from PCR bias, which are the two major sources of errors in variant calling. The reduced computational complexity of mapping selected reads to targeted regions of the genome improves speed, specificity and sensitivity of variant detection. Compared with variant calls from the GATK platform, MiST showed better concordance with SNPs from dbSNP and genotypes determined by an exonic-SNP array. Variant calls made only by MiST confirm at a high rate (>90%) by Sanger sequencing. Thus, MiST is a valuable alternative tool to analyse variants in deep sequencing data.

Diaphragm disease of the small bowel: a retrospective review of CT findings.

OBJECTIVE: The purpose of this article is to report the CT findings of pathologically proven diaphragm disease in the small bowel. MATERIALS AND METHODS: A retrospective review identified 12 patients with pathologically proven small-bowel diaphragm disease who underwent CT within 6 months of surgical resection. Two radiologists, who were unblinded to pathologic and clinical findings, evaluated CT examinations for imaging findings of disease extent, appearance, and location. Clinical history and postoperative follow-up were also performed. RESULTS: The most common presenting symptoms were abdominal pain (7/12 [58%]) and anemia (5/12 [42%]). Long-term use of nonsteroidal antiinflammatory drugs was documented in 58% (7/12) of patients. The most common location of small-bowel diaphragms was the ileum (8/12 [67%]). The CT findings were abnormal in 92% (11/12) of patients. The most common CT findings were small-bowel strictures (11/12 [92%]) and focal (median length, 1 cm) bowel wall thickening (8/12 [67%]). Other less common CT findings included mucosal hyperenhancement (6/12 [50%]), small-bowel dilatation (5/12 [42%]), and video capsule retention (6/9 [67%]). Postoperative follow-up in 11 patients found recurrent symptoms in four patients. CONCLUSION: Small-bowel diaphragm disease should be considered in patients with a history of long-term use of nonsteroidal antiinflammatory drugs, chronic abdominal pain, and anemia who present with CT findings of short, symmetric ileal strictures and focal bowel wall thickening.

Implications of local osteoporosis on the efficacy of anti-resorptive drug treatment: a 3-year follow-up finite element study in risedronate-treated women.

UNLABELLED: The existence of local osteoporosis necessitates patient-specific analysis. Lower and higher ranges of local buckling ratio were found at femoral necks for adequate and inadequate drug response groups, respectively (grouped based on fracture loads). Management of hip fracture risk should be targeted at local geometric abnormalities causing instability. INTRODUCTION: Hip fracture amongst the elderly is a growing concern especially with improvements in living standards and increasing lifespan. Approximately half of the total hip fractures result from those without osteoporosis. This escalates the need to observe local osteoporosis. By observing the local buckling ratio (BR) in the femoral neck in ten risedronate-treated subjects over 3 years, we discovered that subjects with improved fracture loads, as predicted by finite element (FE) analysis, were associated with lower local BR and vice versa. METHODS: The 3D models of the left proximal femurs were generated, and local BR values at 30° intervals were obtained from femoral neck slices by measuring the respective mean cortical thickness and mean outer radius. Following geometric analysis, structural strength was examined with FE analysis where critical fracture loads (F cr) were acquired from sideways fall load simulations. RESULTS: We classified subjects in three groups according to the change in F cr: adequate (+20 %), inadequate (-22 %) and indefinite (-2 %) drug response groups. A common striking feature was that lower and higher ranges of local BR values (baseline year) were found for adequate (min = 2.14, max = 8.04) and inadequate (min = 1.72, max = 11.38) drug response groups, respectively. CONCLUSIONS: Subjects in the inadequate drug response group exhibited high local BR at the supero-anterior and supero-posterior regions. These high local BR values coincided with FE-predicted critical strain regions, whereas subjects from the adequate drug response group showed significantly reduced strain regions. The superiority of coupling geometry (BR) with structure (F cr) over bone mineral density measurements alone by monitoring local osteoporosis has been illustrated.

Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.

Deep sequencing of small RNAs (sRNA-seq) is now the gold standard for small RNA profiling and discovery. Biases in sRNA-seq have been reported, but their etiology remains unidentified. Through a comprehensive series of sRNA-seq experiments, we establish that the predominant cause of the bias is the RNA ligases. We further demonstrate that RNA ligases have strong sequence-specific biases which distort the small RNA profiles considerably. We have devised a pooled adapter strategy to overcome this bias, and validated the method through data derived from microarray and qPCR. In light of our findings, published small RNA profiles, as well as barcoding strategies using adapter-end modifications, may need to be revisited. Importantly, by providing a wide spectrum of substrate for the ligase, the pooled-adapter strategy developed here provides a means to overcome issues of bias, and generate more accurate small RNA profiles.

Marburg and Ebola Virus Infections Elicit a Complex, Muted Inflammatory State in Bats.

The Marburg and Ebola filoviruses cause a severe, often fatal, disease in humans and nonhuman primates but have only subclinical effects in bats, including Egyptian rousettes, which are a natural reservoir of Marburg virus. A fundamental question is why these viruses are highly pathogenic in humans but fail to cause disease in bats. To address this question, we infected one cohort of Egyptian rousette bats with Marburg virus and another cohort with Ebola virus and harvested multiple tissues for mRNA expression analysis. While virus transcripts were found primarily in the liver, principal component analysis (PCA) revealed coordinated changes across multiple tissues. Gene signatures in kidney and liver pointed at induction of vasodilation, reduction in coagulation, and changes in the regulation of iron metabolism. Signatures of immune response detected in spleen and liver indicated a robust anti-inflammatory state signified by macrophages in the M2 state and an active T cell response. The evolutionary divergence between bats and humans of many responsive genes might provide a framework for understanding the differing outcomes upon infection by filoviruses. In this study, we outline multiple interconnected pathways that respond to infection by MARV and EBOV, providing insights into the complexity of the mechanisms that enable bats to resist the disease caused by filoviral infections. The results have the potential to aid in the development of new strategies to effectively mitigate and treat the disease caused by these viruses in humans.

Microencapsulation of probiotics in finger millet milk complex to improve encapsulation efficiency and viability.

This study aimed to assess the effectiveness of finger millet milk complex (almond gum with maltodextrin) to encapsulate the isolated Lactobacillus strains. The wall materials were optimized based on its encapsulation efficiency, antioxidant activity, total phenol content and encapsulation yield. The strains were spray-dried at the optimized condition: 120 °C inlet temperature, maltodextrin 30% and almond gum 1.5%. Survival count of microencapsulated Lactobacillus plantarum RS09 and RS23 strains were 7.91 and 7.83 CFU/g respectively. Viability of microencapsulated strains and free cells under low pH, bile salt, simulated gastric juice and intestinal juice were assessed. Strain RS09 exhibited the highest viable count. Addition of almond gum and finger millet milk increased the phenolic content and offered a protective effect to the strains during spray drying. Results also showed that the powders were amorphous with partial irregularities and a smooth surface with less dents. Hence, they could be used as potential encapsulating agents during spray drying.

Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/µL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.

Effect of the intervertebral disc on vertebral bone strength prediction: a finite-element study.

BACKGROUND CONTEXT: Osteoporotic vertebral fractures (OVFs) are a prevalent skeletal condition in the elderly but the mechanism behind these fractures remain unclear due to the complex biomechanical interplay between spinal segments such as the vertebra and intervertebral discs (IVDs). PURPOSE: To investigate the biomechanical influence of IVDs by (1) comparing finite element (FE)-predicted failure load with experimentally measured failure load of functional spinal units (FSUs) and (2) comparing this correlation with those of FE-predicted failure load and bone mineral density (BMD) of the single central vertebra with experimentally measured failure load. STUDY DESIGN: A computational biomechanical analysis. PATIENT SAMPLE: Ten thoracic FSUs consisting of a central vertebra, the adjacent IVDs, and the upper and lower halves of the adjacent vertebrae were harvested from formalin-fixed human donors (4 males, 6 females; mean age of 82±9 years). OUTCOME MEASURES: The outcome measures included the prediction of vertebral strength and determination of BMD in FSUs and the single central vertebra and the correlation of both measures with experimentally measured vertebral strength of the FSUs. METHODS: The FSUs underwent clinical multidetector computed tomography (MDCT) (spatial resolution: 250×250×600 µm3). BMD was determined for the FSUs from the MDCT images of the central vertebrae. FE-predicted failure load was calculated in the single central vertebra of the FSUs alone and the entire FSUs. Experimentally measured failure load of the FSUs was determined in a uniaxial biomechanical test. RESULTS: BMD of the central vertebrae correlated significantly with experimentally measured failure load (R2=0.66, p<.02), whereas FE-predicted failure load of the central vertebra showed no significant correlation with experimentally measured failure load (p=.07). However, FE-predicted failure load of FSUs best predicted experimentally measured failure load of FSUs (R2=0.93, p<.0001). CONCLUSIONS: This study demonstrated that routine clinical MDCT images can be an accurate and feasible tool for prediction of OVFs using patient-specific FE analysis of FSU models. CLINICAL SIGNIFICANCE: Improved management of OVFs is essential amidst current clinical challenges. Implementation of a vertebral strength assessment tool could result in more accurate prediction of osteoporotic fracture risk and aid clinicians with better targeted early treatment strategies.

Low-dose and sparse sampling MDCT-based femoral bone strength prediction using finite element analysis.

This study aims to evaluate the impact of dose reduction through tube current and sparse sampling on multi-detector computed tomography (MDCT)-based femoral bone strength prediction using finite element (FE) analysis. FE-predicted femoral failure load obtained from MDCT scan data was not significantly affected by 50% dose reductions through sparse sampling. Further decrease in dose through sparse sampling (25% of original projections) and virtually reduced tube current (50% and 25% of the original dose) showed significant effects on the FE-predicted failure load results. PURPOSE: To investigate the effect of virtually reduced tube current and sparse sampling on multi-detector computed tomography (MDCT)-based femoral bone strength prediction using finite element (FE) analysis. METHODS: Routine MDCT data covering the proximal femur of 21 subjects (17 males; 4 females; mean age, 71.0 ± 8.8 years) without any bone diseases aside from osteoporosis were included in this study. Fifty percent and 75% dose reductions were achieved by virtually reducing tube current and by applying a sparse sampling strategy from the raw image data. Images were then reconstructed with a statistically iterative reconstruction algorithm. FE analysis was performed on all reconstructed images and the failure load was calculated. The root mean square coefficient of variation (RMSCV) and coefficient of correlation (R2) were calculated to determine the variation in the FE-predicted failure load data for dose reductions, using original-dose MDCT scan as the standard of reference. RESULTS: Fifty percent dose reduction through sparse sampling showed lower RMSCV and higher correlations when compared with virtually reduced tube current method (RMSCV = 5.70%, R2 = 0.96 vs. RMSCV = 20.78%, R2 = 0.79). Seventy-five percent dose reduction achieved through both methods (RMSCV = 22.38%, R2 = 0.80 for sparse sampling; RMSCV = 24.58%, R2 = 0.73 for reduced tube current) could not predict the failure load accurately. CONCLUSION: Our simulations indicate that up to 50% reduction in radiation dose through sparse sampling can be used for FE-based prediction of femoral failure load. Sparse-sampled MDCT may allow fracture risk prediction and treatment monitoring in osteoporosis with less radiation exposure in the future.

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth.

Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis-like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long-term, multidrug courses with adverse side effects. The emergence of drug-resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next-generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

MDCT-based Finite Element Analysis of Vertebral Fracture Risk: What Dose is Needed?

PURPOSE: The aim of this study was to compare vertebral failure loads, predicted from finite element (FE) analysis of patients with and without osteoporotic vertebral fractures (OVF) at virtually reduced dose levels, compared to standard-dose exposure from multidetector computed tomography (MDCT) imaging and evaluate whether ultra-low dose derived FE analysis can still differentiate patient groups. MATERIALS AND METHODS: An institutional review board (IRB) approval was obtained for this retrospective study. A total of 16 patients were evaluated at standard-dose MDCT; eight with and eight without OVF. Images were reconstructed at virtually reduced dose levels (i. e. half, quarter and tenth of the standard dose). Failure load was determined at L1-3 from FE analysis and compared between standard, half, quarter, and tenth doses and used to differentiate between fracture and control groups. RESULTS: Failure load derived at standard dose (3254 ± 909 N and 3794 ± 984 N) did not significantly differ from half (3390 ± 890 N and 3860 ± 1063 N) and quarter dose (3375 ± 915 N and 3925 ± 990 N) but was significantly higher for one tenth dose (4513 ± 1762 N and 4766 ± 1628 N) for fracture and control groups, respectively. Failure load differed significantly between the two groups at standard, half and quarter doses, but not at tenth dose. Receiver operating characteristic (ROC) curve analysis also demonstrated that standard, half, and quarter doses can significantly differentiate the fracture from the control group. CONCLUSION: The use of MDCT enables a dose reduction of at least 75% compared to standard-dose for an adequate prediction of vertebral failure load based on non-invasive FE analysis.

Feedforward neural network implementation in FPGA using layer multiplexing for effective resource utilization.

This paper presents a hardware implementation of multilayer feedforward neural networks (NN) using reconfigurable field-programmable gate arrays (FPGAs). Despite improvements in FPGA densities, the numerous multipliers in an NN limit the size of the network that can be implemented using a single FPGA, thus making NN applications not viable commercially. The proposed implementation is aimed at reducing resource requirement, without much compromise on the speed, so that a larger NN can be realized on a single chip at a lower cost. The sequential processing of the layers in an NN has been exploited in this paper to implement large NNs using a method of layer multiplexing. Instead of realizing a complete network, only the single largest layer is implemented. The same layer behaves as different layers with the help of a control block. The control block ensures proper functioning by assigning the appropriate inputs, weights, biases, and excitation function of the layer that is currently being computed. Multilayer networks have been implemented using Xilinx FPGA "XCV400hq240". The concept used is shown to be very effective in reducing resource requirements at the cost of a moderate overhead on speed. This implementation is proposed to make NN applications viable in terms of cost and speed for online applications. An NN-based flux estimator is implemented in FPGA and the results obtained are presented.