Bayesian-frequentist hybrid inference framework for single cell RNA-seq analyses.

BACKGROUND: Single cell RNA sequencing technology (scRNA-seq) has been proven useful in understanding cell-specific disease mechanisms. However, identifying genes of interest remains a key challenge. Pseudo-bulk methods that pool scRNA-seq counts in the same biological replicates have been commonly used to identify differentially expressed genes. However, such methods may lack power due to the limited sample size of scRNA-seq datasets, which can be prohibitively expensive. RESULTS: Motivated by this, we proposed to use the Bayesian-frequentist hybrid (BFH) framework to increase the power and we showed in simulated scenario, the proposed BFH would be an optimal method when compared with other popular single cell differential expression methods if both FDR and power were considered. As an example, the method was applied to an idiopathic pulmonary fibrosis (IPF) case study. CONCLUSION: In our IPF example, we demonstrated that with a proper informative prior, the BFH approach identified more genes of interest. Furthermore, these genes were reasonable based on the current knowledge of IPF. Thus, the BFH offers a unique and flexible framework for future scRNA-seq analyses.

Bayesian-frequentist hybrid inference framework for single cell RNA-seq analyses.

Background: Single cell RNA sequencing technology (scRNA-seq) has been proven useful in understanding cell-specific disease mechanisms. However, identifying genes of interest remains a key challenge. Pseudo-bulk methods that pool scRNA-seq counts in the same biological replicates have been commonly used to identify differentially expressed genes. However, such methods may lack power due to the limited sample size of scRNA-seq datasets, which can be prohibitively expensive. Results: Motivated by this, we proposed to use the Bayesian-frequentist hybrid (BFH) framework to increase the power. Conclusion: In our idiopathic pulmonary fibrosis (IPF) case study, we demonstrated that with a proper informative prior, the BFH approach identified more genes of interest. Furthermore, these genes were reasonable based on the current knowledge of IPF. Thus, the BFH offers a unique and flexible framework for future scRNA-seq analyses.

Rift Valley Fever Virus Primes Immune Responses in Aedes aegypti Cells.

The ongoing global emergence of arthropod-borne (arbo) viruses has accelerated research into the interactions of these viruses with the immune systems of their vectors. Only limited information exists on how bunyaviruses, such as Rift Valley fever virus (RVFV), are sensed by mosquito immunity or escape detection. RVFV is a zoonotic phlebovirus (Bunyavirales; Phenuiviridae) of veterinary and human public health and economic importance. We have shown that the infection of mosquitoes with RVFV triggers the activation of RNA interference pathways, which moderately restrict viral replication. Here, we aimed to better understand the interactions between RVFV and other vector immune signaling pathways that might influence RVFV replication and transmission. For this, we used the immunocompetent Aedes aegypti Aag2 cell line as a model. We found that bacteria-induced immune responses restricted RVFV replication. However, virus infection alone did not alter the gene expression levels of immune effectors. Instead, it resulted in the marked enhancement of immune responses to subsequent bacterial stimulation. The gene expression levels of several mosquito immune pattern recognition receptors were altered by RVFV infection, which may contribute to this immune priming. Our findings imply that there is a complex interplay between RVFV and mosquito immunity that could be targeted in disease prevention strategies.

Enhanced Nanobubble Formation: Gold Nanoparticle Conjugation to Qß Virus-like Particles.

Plasmonic gold nanostructures are a prevalent tool in modern hypersensitive analytical techniques such as photoablation, bioimaging, and biosensing. Recent studies have shown that gold nanostructures generate transient nanobubbles through localized heating and have been found in various biomedical applications. However, the current method of plasmonic nanoparticle cavitation events has several disadvantages, specifically including small metal nanostructures (≤10 nm) which lack size control, tuneability, and tissue localization by use of ultrashort pulses (ns, ps) and high-energy lasers which can result in tissue and cellular damage. This research investigates a method to immobilize sub-10 nm AuNPs (3.5 and 5 nm) onto a chemically modified thiol-rich surface of Qß virus-like particles. These findings demonstrate that the multivalent display of sub-10 nm gold nanoparticles (AuNPs) caused a profound and disproportionate increase in photocavitation by upward of 5-7-fold and significantly lowered the laser fluency by 4-fold when compared to individual sub-10 nm AuNPs. Furthermore, computational modeling showed that the cooling time of QßAuNP scaffolds is significantly extended than that of individual AuNPs, proving greater control of laser fluency and nanobubble generation as seen in the experimental data. Ultimately, these findings showed how QßAuNP composites are more effective at nanobubble generation than current methods of plasmonic nanoparticle cavitation.

Chemical and biology of sulfur fluoride exchange (SuFEx) click chemistry for drug discovery.

Compounds containing an SF bond have garnered intense interest in the chemical and biological literature. In particular, sulfonyl fluorides (RSO2F) are commonly used as covalent protein inhibitors and biological probes. The introduction of the fluorine atom into drugs often leads to significantly promoted medicinal properties, which revolutionized the development of pharmaceuticals and gained attention because of the beneficial properties of these small and highly electro-negative halogens. The sulfonyl fluoride functional group has also been widely adopted throughout the field of chemical biology due to its unique balance between reactivity and stability under physiological conditions. This comprehensive review highlights the recent developments of sulfonyl fluorides based compounds in a massive range of therapeutic applications. We believe this review article will be helpful to inspire new ideas for structural design and developments of less toxic and potent Sulfur based drugs against the numerous death-causing diseases.

QRS fragmentation and prolongation as predictors of exercise capacity in patients after Fontan palliation.

INTRODUCTION: Patients with Fontan anatomy are at increased risk for exercise intolerance and early morbidity and mortality. QRS complex fragmentation (fQRS) and prolongation have been studied in multiple heart diseases, but their clinical importance is unknown in the Fontan population. METHODS: A retrospective cross-sectional study was performed. ECGs were evaluated for QRS prolongation (>98 percentile for age) and fQRS (≥3 R-waves/notches in the R/S complex [more than two in RBBB] in ≥2 contiguous leads). The primary outcome measures were CPET performance. RESULTS: Total 90 patients (median age 18 years, 57% male, 59% RV dominant) were included; 13% had fQRS and 31% had prolonged QRS. Demographically, patients with fQRS or prolonged QRS were like those without. Peak VO2 (64% vs. 63%, p .45), VE/VCO2 slope (85% vs. 88%, p = .74), and O2 pulse (149% vs. 129%, p = .83) were similar in the fQRS group versus those without. Upon multi-variable regression, body mass index (ß = -0.38, p < .01) and QRS duration (ß = -0.29, p < .01) were independently associated with % predicted VO2; fQRS was not. Lower cardiac index (2.2 vs. 2.8 L/min/m2 , p = .03) and higher ventricular end-diastolic pressure (13 vs. 10 mmHg, p = .02) was seen with fQRS. CONCLUSIONS: QRS fragmentation is present in patients with Fontan physiology. fQRS showed no association with CPET performance but was related to invasive hemodynamic markers of ventricular performance. QRS duration may be a better predictor of exercise function following Fontan.

Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6-7 linker.

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate-proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236-265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6-7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6-7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1-6 and TMD7-12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252-265 from TMD7-12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6-7 loop (positions 236-250 or 251-265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT-ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6-7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6.

The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G(93)XXXG(97)) and TMD4 (G(155)XXXG(159)). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [(3)H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bismethanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4.

Distal junctional kyphosis of adolescent idiopathic thoracic curves following anterior or posterior instrumented fusion: incidence, risk factors, and prevention.

STUDY DESIGN: This is a retrospective multicenter analysis of a subset of 375 patients with thoracic adolescent idiopathic scoliosis (AIS) treated with either anterior (238) or posterior (137) fusion with preoperative or postoperative distal junctional kyphosis (DJK) >or=10 degrees . OBJECTIVES: To determine the incidence of DJK before and after surgery in patients with AIS undergoing either anterior or posterior thoracic fusion, and provide recommendations for prevention. SUMMARY OF BACKGROUND DATA: DJK following surgical treatment for AIS may result in pain, imbalance, and unacceptable deformity. The true incidence of DJK following selective anterior or posterior instrumentation and fusion is unknown, as are "risk factors" for its development. METHODS: Mean age at surgery was 14.4 years (range 9.1-20.9) in the anterior group and 14.7 years (range 10.2-20.7) in the posterior. Analysis included the Cobb and instrumented levels of the thoracic curves, and sagittal measurements, all on preoperative and 2-year follow-up standing 36-in radiographs. RESULTS: In the anterior group, the incidence of preoperative DJK was 4.2%, and postoperative DJK was 7.1%. In the posterior group, the incidence of preoperative DJK was 5.0% and 14.6% after surgery. When postoperative DJK developed in the posterior group, mean postoperative T10-L2 was +17 degrees kyphosis compared to +2 degrees in the posterior group without DJK (P < 0.001). When postoperative DJK developed in the anterior group, mean postoperative T10-L2 was +12 degrees kyphosis compared to +2 degrees for the anterior group without DJK (P = 0.006). DJK was significantly more likely to occur in the posterior group if the Cobb was instrumented to less than Cobb +1 (P < 0.001). CONCLUSIONS: It appears that both posterior and anterior instrumentation for thoracic curves must include the junctional level to prevent postoperative DJK when postoperative DJK is present. The presence of increased kyphosis after surgery in the T10-L2 region seen in both anterior and posterior groups that had postoperative DJK develop constitutes a "risk factor" for the development of DJK.

A posterior tether for fusionless modulation of sagittal plane growth in a sheep model.

STUDY DESIGN: This was an in vivo and in vitro investigation of a minimally invasive posterior tether system for fusionless modulation of sagittal plane growth in an immature sheep model. OBJECTIVE: To determine whether fusionless modulation of spinal growth in the sagittal plane of an immature sheep model can be successfully performed. SUMMARY OF BACKGROUND DATA: Scheuermann's disease has been reported to affect 1% to 8% of the pediatric population. Varying degrees of spinal bone growth modulation has been successfully performed in the coronal plane of animal models. This represents the first attempt to modulate spinal growth in the sagittal plane in an animal model. Modulation of spinal growth in the sagittal plane may play a role in the treatment of pediatric kyphosis when brace treatment is unsuccessful. METHODS: There were nine immature sheep posteriorly tethered and five control animals, all with identical follow-ups. Pedicle screws were placed in adjacent vertebrae, bilaterally, at the thoracolumbar junction and the lower lumbar spine. Polyethylene cords were passed subcutaneously, and tensioned to 20 lbs. Tethers were released in four sheep at 6 months and four at 12 months. At 12 months and 2 weeks, all animals were sacrificed. RESULTS: At 13 months after surgery, the tethered groups had significantly less kyphosis and vertebral body wedging than the control group. In both the tethered groups, at 13 months after surgery, there was significantly less vertebral body wedging compared with the preoperative values. There was no evidence of facet arthropathy, but heterotopic ossification was seen in several specimens. Tethered spines had significantly less motion than control spines. Histologic evaluation demonstrated no detrimental results. CONCLUSIONS: Fusionless modulation of sagittal plane growth in an immature sheep model was successfully performed with changes in the overall sagittal profile of the instrumented segments and vertebral body wedging. Increased stiffness of the instrumented segments posttether release was thought to be related to heterotopic ossification. Results suggest that this may be a potential treatment for adolescents with Scheuermann's disease.

Single-rod versus dual-rod anterior instrumentation for idiopathic scoliosis: a biomechanical study.

STUDY DESIGN: Anterior single- and dual-rod instrumented human and ovine thoracolumbar spines, with and without structural interbody support (SIS), were biomechanically tested and compared in flexion, lateral bending, and torsion. OBJECTIVE: To determine significant differences in global stiffness of the constructs in an attempt to clarify specific indications for each in the treatment of spinal deformities. SUMMARY OF BACKGROUND DATA: Single- and dual-rod anterior systems have been used without any consensus as to indications for one versus the other. The potential added benefit of incorporating SIS and transverse connectors (dual-rod) with these constructs has also not been fully explored. METHODS: Four human cadaveric and six ovine spines were instrumented in single- and dual-rod constructs and biomechanically tested intact, postdiscectomy with and without SIS, with single- and dual-rod constructs, and with and without transverse connectors (ovine only). Biomechanical testing modes were flexion, lateral bending, and torsion. RESULTS: In the human cadaveric specimens, testing in flexion revealed that SIS was the major contributing factor for construct stiffness. In lateral bending, stiffness of single- and dual-rod constructs with and without SIS was equivalent. In torsion, both single- and dual-rod instrumentation and SIS appeared to contribute to global stiffness. In ovine specimens, dual rods were stiffer than single-rod constructs and SIS played only a minor role. Transverse connectors appeared to significantly stiffen dual-rod constructs in torsion only. CONCLUSIONS: Dual-rod constructs with SIS appear to be the best combination for providing stiffness in anterior instrumentation. The addition of cross-links to anterior constructs does not appear to increase stiffness except in torsion.

A biomechanical study of regional endplate strength and cage morphology as it relates to structural interbody support.

STUDY DESIGN: An in vitro biomechanical investigation to quantify the endplates resistance to compressive loads, in the thoracic and lumbar spine. Comparisons were made to determine the regional strength of the endplate, the optimal size and geometry of interbody support, and the effects of endplate removal on structural strength. OBJECTIVES: To biomechanically assess the regional variation of endplate strength in the thoracic and lumbar spine, the optimal geometry and cross-sectional area for structural interbody support, and endplate preparation techniques with respect to endplate failure or subsidence. SUMMARY OF BACKGROUND DATA: Anterior column interbody support plays an important role in spinal reconstruction. Subsidence of interbody structural support is a common problem and may be related to regional weakness of the endplate, the size and/or geometry of structural support, and the preparation of the endplate. Biomechanical data related to these issues should be of importance to spine surgeons and reduce the risk of subsidence and its inherent complications. METHODS: The indentation tests were performed in three subgroups, each with a different set of test variables. The first test consisted of 65 vertebrae at six different endplate test positions using a 9.53-mm diameter indenter. The second test was performed on 48 vertebrae at a central endplate test site using three hollow and two solid cylindrical indenters of varying diameter. The third test was done using 24 vertebrae with the endplate intact, partially removed, or fully removed. All tests were run using human cadaveric specimen using both the superior and inferior endplates. The maximum load to failure (MLF) was determined for each test performed. RESULTS: For all levels tested, the highest MLF occurred in the posterolateral region of the endplate. The lowest value occurred in the central and anterocentral regions for levels T7-L5 and T1-T6, respectively. Hollow indenters with a small diameter had the lowest MLF, whereas solid large-diameter indenters had the highest MLF. The ultimate compressive strength for all hollow indenters was significantly higher than all solid indenters. There was a significant reduction in the endplate strength with the complete removal of the endplate. CONCLUSIONS: The posterolateral region of the endplate provides the greatest resistance to subsidence while the central region provides the least resistance. A larger-diameter solid support has the greater MLF and the lower the risk of subsidence, suggesting a more efficient transfer of force to the endplate with the hollow indenters. Parameters such as the geometry of structural support and the position and preparation of the endplate can influence the resistance of an interbody support to subside. Partial removal of the endplate may provide both, for adequate mechanical advantage and a highly vascular site for fusion.