IsoVis - a webserver for visualization and annotation of alternative RNA isoforms.

Genes commonly express multiple RNA products (RNA isoforms), which differ in exonic content and can have different functions. Making sense of the plethora of known and novel RNA isoforms being identified by transcriptomic approaches requires a user-friendly way to visualize gene isoforms and how they differ in exonic content, expression levels and potential functions. Here we introduce IsoVis, a freely available webserver that accepts user-supplied transcriptomic data and visualizes the expressed isoforms in a clear, intuitive manner. IsoVis contains numerous features, including the ability to visualize all RNA isoforms of a gene and their expression levels; the annotation of known isoforms from external databases; mapping of protein domains and features to exons, allowing changes to protein sequence and function between isoforms to be established; and extensive species compatibility. Datasets visualised on IsoVis remain private to the user, allowing analysis of sensitive data. IsoVis visualisations can be downloaded to create publication-ready figures. The IsoVis webserver enables researchers to perform isoform analyses without requiring programming skills, is free to use, and available at https://isomix.org/isovis/.

Control of Neuronal Survival and Development Using Conductive Diamond.

This study demonstrates the control of neuronal survival and development using nitrogen-doped ultrananocrystalline diamond (N-UNCD). We highlight the role of N-UNCD in regulating neuronal activity via near-infrared illumination, demonstrating the generation of stable photocurrents that enhance neuronal survival and neurite outgrowth and foster a more active, synchronized neuronal network. Whole transcriptome RNA sequencing reveals that diamond substrates improve cellular-substrate interaction by upregulating extracellular matrix and gap junction-related genes. Our findings underscore the potential of conductive diamond as a robust and biocompatible platform for noninvasive and effective neural tissue engineering.

3D-printed low-cost choke corrugated Gaussian profile horn antenna for Ka-band.

In this work, a fully 3D-printed choke corrugated Gaussian profile horn antenna (GPHA) using high-conductive filaments and a low-cost modular 3D-printing technique is implemented. The choke corrugated GPHA operates in the Ka-band, with a central frequency of 28 GHz. Although the antenna can be printed in one piece as its dimensions are within the printing limits, four pieces compose the three sections of the final 3D-printed antenna. The numerical simulations and measurements of the antenna show a good agreement, validating the possibility of cost-effective modular fabrication of this complex type of antennas.

Pervasive effects of RNA degradation on Nanopore direct RNA sequencing.

Oxford Nanopore direct RNA sequencing (DRS) is capable of sequencing complete RNA molecules and accurately measuring gene and isoform expression. However, as DRS is designed to profile intact RNA, expression quantification may be more heavily dependent upon RNA integrity than alternative RNA sequencing methodologies. It is currently unclear how RNA degradation impacts DRS or whether it can be corrected for. To assess the impact of RNA integrity on DRS, we performed a degradation time series using SH-SY5Y neuroblastoma cells. Our results demonstrate that degradation is a significant and pervasive factor that can bias DRS measurements, including a reduction in library complexity resulting in an overrepresentation of short genes and isoforms. Degradation also biases differential expression analyses; however, we find that explicit correction can almost fully recover meaningful biological signal. In addition, DRS provided less biased profiling of partially degraded samples than Nanopore PCR-cDNA sequencing. Overall, we find that samples with RNA integrity number (RIN) > 9.5 can be treated as undegraded and samples with RIN > 7 can be utilized for DRS with appropriate correction. These results establish the suitability of DRS for a wide range of samples, including partially degraded in vivo clinical and post-mortem samples, while limiting the confounding effect of degradation on expression quantification.

Identification of cell barcodes from long-read single-cell RNA-seq with BLAZE.

Long-read single-cell RNA sequencing (scRNA-seq) enables the quantification of RNA isoforms in individual cells. However, long-read scRNA-seq using the Oxford Nanopore platform has largely relied upon matched short-read data to identify cell barcodes. We introduce BLAZE, which accurately and efficiently identifies 10x cell barcodes using only nanopore long-read scRNA-seq data. BLAZE outperforms the existing tools and provides an accurate representation of the cells present in long-read scRNA-seq when compared to matched short reads. BLAZE simplifies long-read scRNA-seq while improving the results, is compatible with downstream tools accepting a cell barcode file, and is available at https://github.com/shimlab/BLAZE .

Arthroscopic Lysis of Adhesions for the Stiff Total Knee Arthroplasty.

Background: Arthroscopic lysis of adhesions is a treatment option for patients with painful, stiff knees as a result of arthrofibrosis following knee arthroplasty, in whom prior manipulation under anesthesia (MUA) has failed. Typically, nonoperative treatment in these patients has also failed, including aggressive physiotherapy, stretching, dynamic splinting, and various pain-management measures or medications. Range of motion in these patients is often suboptimal, and any gains in flexibility will likely have hit a plateau over many months. The goal of performing lysis of adhesions is to increase the range of motion in patients with knee stiffness following total knee arthroplasty, as well as to reduce pain and restore physiologic function of the knee, enabling activities of daily living. Description: This is a straightforward surgical technique that can be performed in a single stage. The preoperative range of motion is documented after induction of general anesthesia. The procedure begins with the establishment of standard medial and lateral parapatellar arthroscopic portals. A blunt trocar is introduced into the knee, and blunt, manual lysis of adhesions is performed in the suprapatellar pouch and the medial and lateral gutters with use of a sweeping motion after piercing and perforating the scarred adhesive bands or capsular tissue. Next, the arthroscope is inserted into the knee, and a diagnostic arthroscopy is performed. Bands of fibrous tissue are released and resected with use of electrocautery and a 4.0-mm arthroscopic shaver. Next, the posterior cruciate ligament (PCL) is visualized in full flexion. If PCL tightness is observed, the PCL can be released from its femoral origin until the flexion gap is increased. This portion of the procedure can include either partial or full release of the PCL, as indicated. Next, the arthroscope is removed and the ipsilateral hip is flexed to 90° for a standard MUA. Gentle force is applied to the proximal aspect of the tibia, and the knee is flexed. After completing the MUA, immediate post-intervention range of motion of the knee is documented, and the patient is provided with a continuous passive motion (CPM) machine set to the maximum flexion and extension achieved in the operating room. Alternatives: Nonoperative treatment of a stiff knee following total knee arthroplasty is well documented in the current literature. Range of motion has been shown to increase in patients undergoing proper pain management, aggressive physical therapy, and closed MUA in the acute postoperative setting. Additionally, more severe cases of established arthrofibrosis despite prior MUA can be treated with an open lysis of adhesions1-3. Rationale: Arthroscopic lysis of adhesions with PCL release versus resection has been well described previously. This procedure has been shown to benefit patients in whom initial nonoperative treatment has failed. Additionally, this procedure is not limited to the immediate acute postoperative period like standard MUA3. To our knowledge, no technique video has been published outlining arthroscopic lysis of adhesions for a stiff knee following total knee arthroplasty. Expected Outcomes: This procedure has been shown to provide an immediate and lasting improvement in the flexion and extension arc of the knee, as well as improved functional outcomes. Patients should be educated that improvements gained in the operating room must be sustained through physical therapy. In a study of 32 patients who underwent arthroscopic lysis of adhesions for moderately severe arthrofibrosis following a total knee arthroplasty, Jerosch and Aldawoudy reported a mean postoperative flexion of 119° in the operating room and 97° at the time of the latest follow-up. Eight patients with extensor lag showed improvement from 27° to 4°. Average Knee Society scores improved from 70 points preoperatively to 86 points at the time of the latest follow-up4. Their article showed that arthroscopic treatment of stiffness following total knee arthroplasty is a safe and effective form of treatment. Important Tips: Perform manual lysis of adhesions with a trocar prior to inserting the arthroscope in order to improve visualization and access.Utilize all portals and accessory portals interchangeably in order to improve access.Prescribe physical therapy with or without CPM machine immediately following surgery in order to maintain correction.Utilize pump inflow in order to help distend the tightened capsule.Protect the prosthetic surface from scratches during portal establishment.Loss of flexion implies scarring in the suprapatellar pouch and/or intercondylar notch, or PCL tightness.Loss of extension implies a tight posterior capsule, posterior osteophytes, or scarring of the PCL stump.A motorized shaver is the best tool for treatment of dense fibrous tissue, but be sure not to scratch metal total knee components. Acronyms and Abbreviations: TKA = total knee arthroplastyROM = range of motionCT = computed tomographyMRI = magnetic resonance imagingESR = erythrocyte sedimentation rateCRP = C-reactive protein.

Synergistic effects of robotic surgery and IPACK nerve block on reduction of opioid consumption in total knee arthroplasty.

Background: There are numerous strategies to combat postoperative analgesia and expedite recovery after total knee arthroplasty (TKA). The purpose of this study was to determine opioid consumption, length of stay, and functional outcomes after robotic versus standard TKA in the setting of various regional pain modalities. Methods: A consecutive series of patients treated with unilateral primary robotic or standard TKA from January 2018-February 2021 were retrospectively identified. Regional pain modalities included peri-articular injection (PAI), adductor canal block (ACB), and infiltration between popliteal artery and capsule of knee (IPACK). Patient demographics, operative/perioperative variables, and postoperative function were recorded. Daily opiate consumption was calculated as morphine milligram equivalents (MME). Multivariate regression was performed to control for age, sex, and race. Results: After review, 283 patients (177 Females; 106 Males) were included. Robotic TKA patients received IPACK + ACB (36), while standard TKA patients received either ACB (45), IPACK + ACB (167), or PAI (35). Daily inpatient opioid consumption in the standard IPACK + ACB (p = 0.02) and robotic IPACK + ACB groups (p = 0.0001) was significantly lower compared to standard ACB. When combined with IPACK block, robotic procedures synergistically lowered opiate consumption (p = 0.004) compared to standard procedures and led to earlier discharge (p = 0.003). The robotic IPACK + ACB cohort also demonstrated improved early ambulation compared to standard ACB, (p = 0.05), whereas the same benefit was not seen for patients who received IPACK during standard TKA. Conclusions: The utilization of IPACK block decreases inpatient postoperative opioid requirements following TKA. Robotic TKA and IPACK block appeared to have a synergistic effect on opioid consumption and postoperative recovery.

Accurate expression quantification from nanopore direct RNA sequencing with NanoCount.

Accurately quantifying gene and isoform expression changes is essential to understanding cell functions, differentiation and disease. Sequencing full-length native RNAs using long-read direct RNA sequencing (DRS) has the potential to overcome many limitations of short and long-read sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. However, there are a lack of tools specifically designed for DRS and its ability to identify differential expression in complex organisms is poorly characterised. We developed NanoCount for fast, accurate transcript isoform quantification in DRS and demonstrate it outperforms similar methods. Using synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that DRS accurately quantifies RNA expression and identifies differential expression of genes and isoforms. Differential expression of 231 genes, 333 isoforms, plus 27 isoform switches were detected between undifferentiated and differentiated SH-SY5Y cells and samples clustered by differentiation state at the gene and isoform level. Genes upregulated in neuron-like cells were associated with neurogenesis. NanoCount quantification of thousands of novel isoforms discovered with DRS likewise enabled identification of their differential expression. Our results demonstrate enhanced DRS isoform quantification with NanoCount and establish the ability of DRS to identify biologically relevant differential expression of genes and isoforms.

Learning curve for active robotic total knee arthroplasty.

PURPOSE: Total Knee Arthroplasty (TKA) procedures incorporate technology in an attempt to improve outcomes. The Active Robot (ARo) performs a TKA with automated resections of the tibia and femur in efforts to optimize bone cuts. Evaluating the Learning Curve (LC) is essential with a novel tool. The purpose of this study was to assess the associated LC of ARo for TKA. METHODS: A multi-center prospective FDA cohort study was conducted from 2017 to 2018 including 115 patients that underwent ARo. Surgical time of the ARo was defined as Operative time (OT), segmented as surgeon-dependent time (patient preparation and registration) and surgeon-independent time (autonomous bone resection by the ARo). An average LC for all surgeons was computed. Complication rates and patient-reported outcome (PRO) scores were recorded and examined to evaluate for any LC trends in these patient related factors. RESULTS: The OT for the cases 10-12 were significantly quicker than the OT time of cases 1-3 (p < 0.028), at 36.5 ± 7.4 down from 49.1 ± 17 min. CUSUM and confidence interval analysis of the surgeon-dependent time showed different LCs for each surgeon, ranging from 12 to 19 cases. There was no difference in device related complications or PRO scores over the study timeframe. CONCLUSION: Active Robotic total knee arthroplasty is associated with a short learning curve of 10-20 cases. The learning curve was associated with the surgical time dedicated to the robotic specific portion of the case. There was no learning curve-associated device-related complications, three-dimensional component position, or patient-reported outcome scores. LEVEL OF EVIDENCE: Level II.

Safe and effective use of active robotics for TKA: Early results of a multicenter study.

BACKGROUND: A novel active robotic system for total knee arthroplasty (TKA) performs automated milling of bone surfaces. Study objectives were to assess system safety and effectiveness in a US population. METHODS: A multicenter clinical trial was conducted, following 115 patients for at least 6-months. A pre-defined list of robot-related adverse events was used to evaluate safety. Efficacy was assessed radiographically comparing planned versus achieved coronal limb alignment. RESULTS: No pre-defined adverse events occurred and postoperative limb alignment more than ±3° from plan occurred in 11.2 % of cases. CONCLUSION: Active robotics for TKA is safe and effective as demonstrated in this trial.

Major SCP/TAPS protein expansion in Lucilia cuprina is associated with novel tandem array organisation and domain architecture.

BACKGROUND: Larvae of the Australian sheep blowfly, Lucilia cuprina, parasitise sheep by feeding on skin excretions, dermal tissue and blood, causing severe damage known as flystrike or myiasis. Recent advances in -omic technologies and bioinformatic data analyses have led to a greater understanding of blowfly biology and should allow the identification of protein families involved in host-parasite interactions and disease. Current literature suggests that proteins of the SCP (Sperm-Coating Protein)/TAPS (Tpx-1/Ag5/PR-1/Sc7) (SCP/TAPS) superfamily play key roles in immune modulation, cross-talk between parasite and host as well as developmental and reproductive processes in parasites. METHODS: Here, we employed a bioinformatics workflow to curate the SCP/TAPS protein gene family in L. cuprina. Protein sequence, the presence and number of conserved CAP-domains and phylogeny were used to group identified SCP/TAPS proteins; these were compared to those found in Drosophila melanogaster to make functional predictions. In addition, transcription levels of SCP/TAPS protein-encoding genes were explored in different developmental stages. RESULTS: A total of 27 genes were identified as belonging to the SCP/TAPS gene family: encoding 26 single-domain proteins each with a single CAP domain and a solitary double-domain protein containing two conserved cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domains. Surprisingly, 16 SCP/TAPS predicted proteins formed an extended tandem array spanning a 53 kb region of one genomic region, which was confirmed by MinION long-read sequencing. RNA-seq data indicated that these 16 genes are highly transcribed in all developmental stages (excluding the embryo). CONCLUSIONS: Future work should assess the potential of selected SCP/TAPS proteins as novel targets for the control of L. cuprina and related parasitic flies of major socioeconomic importance.

Active Robotic Total Knee Arthroplasty (TKA): Initial Experience with the TSolution One ® TKA System.

Several recent advances, including the use of robotic devices, have been explored to improve outcomes in total knee arthroplasty (TKA). The TSolution One ® Total Knee Application (THINK Surgical, Inc., Fremont, CA, USA) introduces an active robotic device that supports an open implant platform and CT-based preoperative planning workflow, and requires minimal surgeon intervention for making bone cuts. Our experience was part of a multi-center, prospective, non-randomized trial assessing the safety and effectiveness of this active robotic system for TKA. Each patient underwent a preoperative CT-scan, which was uploaded to proprietary planning software. The surgeon reviewed the software-generated 3D digital model, selected the appropriate implants and generated a final preoperative plan. Intra-operatively, a standard medial parapatellar approach was used. The leg was then rigidly attached to the robot via fixation pins, and registration markers were placed in the tibia and femur. Landmark registration was performed to inform the robot of the knee's position in space and to confirm the robot's ability to execute the preoperative plan. Next, the robot performed femoral and tibial cuts using a cutter in a sequential fashion along a defined cut-path. The robot was then removed from the operative field and the surgeon completed the procedure by removing marginal bone and performing final balancing and implantation in the usual fashion. The TSolution One® Total Knee Application is a computer-assisted device that potentially allows a surgeon to make more accurate cuts and to determine optimal implant position based on the patient's specific anatomy. It is the only active robotic system currently available. In this manuscript, we describe the operative technique and workflow involved in performing this surgery and offer insight on optimizing safety and efficiency as we introduce new technologies to the operating theater. We also present two cases performed by the senior author to further demonstrate technical aspects of the procedure.

Incidental Asymptomatic Fibular Stress Fractures Presenting as Varus Knee Osteoarthritis: A Case Report.

Stress fractures are often missed, especially in unusual clinical settings. We report on 2 patients who presented to our orthopedic surgery clinic with incidental findings of asymptomatic proximal fibular tension side stress fractures in severe longstanding varus osteoarthritic knees. Initial plain films demonstrated an expansile deformity of the proximal fibular shaft, and differential diagnosis included a healed or healing fracture versus possible neoplasm. Magnetic resonance imaging with and without gadolinium was utilized to rule out the latter prior to planned total knee arthroplasty.

Ticlopidine-induced cholestatic hepatitis.

OBJECTIVE: To report 2 cases of ticlopidine-induced cholestatic hepatitis, investigate its mechanism, and compare the observed main characteristics with those of the published cases. CASE SUMMARIES: Two patients developed prolonged cholestatic hepatitis after receiving ticlopidine following percutaneous coronary angioplasty, with complete remission during the follow-up period. T-cell stimulation by therapeutic concentration of ticlopidine was demonstrated in vitro in the patients, but not in healthy controls. DISCUSSION: Cholestatic hepatitis is a rare complication of the antiplatelet agent ticlopidine; several cases have been reported but few in the English literature. Our patients developed jaundice following treatment with ticlopidine and showed the clinical and laboratory characteristics of cholestatic hepatitis, which resolved after discontinuation of the drug. Hepatitis may develop weeks after discontinuation of the drug and may run a prolonged course, but complete remission was observed in all reported cases. An objective causality assessment revealed that the adverse drug event was probably related to the use of ticlopidine. The mechanisms of this ticlopidine-induced cholestasis are unclear. Immune mechanisms may be involved in the drug's hepatotoxicity, as suggested by the T-cell stimulation study reported here. CONCLUSIONS: Cholestatic hepatitis is a rare adverse effect of ticlopidine that may be immune mediated. Patients receiving the drug should be monitored with liver function tests along with complete blood cell counts. This complication will be observed even less often in the future as ticlopidine is being replaced by the newer antiplatelet agent clopidogrel.

Serum levels of inhibin B, unlike inhibin A and activin A, are not altered in women with preeclampsia.

PROBLEM: To investigate whether inhibin A, inhibin B, and activin A serum levels are altered in women with preeclampsia. METHOD OF STUDY: Serum samples of 20 women with preeclampsia (study group) and 20 normotensive pregnant women, matched for maternal and gestational age and parity, were assayed for inhibin A, inhibin B and activin A by specific enzyme-linked immunosorbent assay. RESULTS: Median serum concentrations of inhibin A and activin A were significantly higher among women with preeclampsia than in women with normotensive pregnancies, while inhibin B levels were comparable in both groups. Activin A levels were positively correlated with those of inhibins A and B, and inhibin A levels were positively correlated with diastolic blood pressure and inhibin B concentration in the study group. CONCLUSIONS: Inhibin A and activin A, but not inhibin B, serum levels are markedly increased in women with preeclampsia. These hormones might serve as an endocrine marker for preeclampsia.

Low-molecular-weight heparin for the prevention of obstetric complications in women with thrombophilias.

OBJECTIVE: To evaluate the benefit of combined low-molecular-weight (LMW) heparin and aspirin for prophylaxis in women carriers of thrombophilia who had previously suffered from severe obstetric complications. METHODS: The 33 studied women had an earlier pregnancy complicated by severe preeclampsia, abruptio placentae, intrauterine growth retardation, or intrauterine fetal death. All were subsequently diagnosed as carrying inherited thrombophilias. In their subsequent pregnancy, prophylactic therapy consisting of LMW heparin 40 mg/day (Enoxaparin, Rhone-Poulenc-Rorer, France) and aspirin was administered. Patients who were found to be homozygotes for the methylenetetrahydrofolate reductase mutation also received folic acid supplementation throughout their pregnancy. RESULTS: Low-molecular-weight heparin was well tolerated and none of the women or the newborns developed any hemorrhagic complications. Only three (9.1%) of the women developed pregnancy complications. The mean gestational age and the mean birth weight at delivery in the previously complicated pregnancies were 32.1 +/- 5.0 weeks and 1175 +/- 590 g, respectively, compared to 37.6 +/- 2.3 weeks and 2719 +/- 526 g, respectively, in the treated pregnancies (p < 0.001). CONCLUSIONS: This uncontrolled trial suggests that patients with obstetric complications and an inherited thrombophilia may benefit from treatment with combined LMW heparin and aspirin in subsequent pregnancies. However, this needs to be verified by controlled trials before considering clinical application.

p53 and WAF1 polymorphisms in Jewish-Israeli women with epithelial ovarian cancer and its association with BRCA mutations.

OBJECTIVE: To investigate whether polymorphic p53 and WAF1 alleles are associated with clinical, demographic and histopathological features and BRCA mutation in women with ovarian cancer. DESIGN: A cross-sectional study. POPULATION: Two hundred and twenty-one nonselected Israeli women with epithelial ovarian cancer. METHODS: DNA was analysed for known polymorphisms in intron 3 (a 16 nucleotide single repeat) and intron 6 (a G to A change at nucleotide 13,494) of the p53 gene, the S31R polymorphism in the WAF1 gene, and for three predominant Jewish mutations in the BRCA genes (185delAG and 5382insC in BRCA1, and 6174delT in BRCA2). MAIN OUTCOME MEASURE: The rate of polymorphic p53 and WAF1 alleles and their association with BRCA mutation, ethnic origin, age and stage at diagnosis, and family history of cancer. RESULTS: Of the tested women, 72 (32.6%) were either BRCA1 (n = 57) or BRCA2 (n = 15) mutation carriers. Sixty-eight of 213 (31.9%) were heterozygous for intron 3 polymorphism, 67/193 (34.7%) for intron 6 polymorphism, and 22/154 (14.3%) for S31R of the WAF1 gene. The p53 and WAF1 polymorphism rate did not differ between BRCA mutation carriers and noncarriers. No significant association between specific p53 or WAF1 genotypes, and clinical, histopathological or demographic variables was observed. CONCLUSION: In Jewish-Israeli women with sporadic and familial ovarian cancer, p53 or WAF1 polymorphisms do not seem to affect the phenotype.