Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Depression after stoma surgery: a systematic review and meta-analysis.

BACKGROUND: Depression is the leading cause of global disability and can develop following the change in body image and functional capacity associated with stoma surgery. However, reported prevalence across the literature is unknown. Accordingly, we performed a systematic review and meta-analysis aiming to characterise depressive symptoms after stoma surgery and potential predictive factors. METHODS: PubMed/MEDLINE, Embase, CINAHL and Cochrane Library were searched from respective database inception to 6 March 2023 for studies reporting rates of depressive symptoms after stoma surgery. Risk of bias was assessed using the Downs and Black checklist for non-randomised studies of interventions (NRSIs), and Cochrane RoB2 tool for randomised controlled trials (RCTs). Meta-analysis incorporated meta-regressions and a random-effects model. REGISTRATION: PROSPERO, CRD42021262345. RESULTS: From 5,742 records, 68 studies were included. According to Downs and Black checklist, the 65 NRSIs were of low to moderate methodological quality. According to Cochrane RoB2, the three RCTs ranged from low risk of bias to some concerns of bias. Thirty-eight studies reported rates of depressive symptoms after stoma surgery as a proportion of the respective study populations, and from these, the median rate across all timepoints was 42.9% 42.9% (IQR: 24.2-58.9%). Pooled scores for respective validated depression measures (Hospital Anxiety and Depression Score (HADS), Beck Depression Inventory (BDI), and Patient Health Questionnaire-9 (PHQ-9)) across studies reporting those scores were below clinical thresholds for major depressive disorder according to severity criteria of the respective scores. In the three studies that used the HADS to compare non-stoma versus stoma surgical populations, depressive symptoms were 58% less frequent in non-stoma populations. Region (Asia-Pacific; Europe; Middle East/Africa; North America) was significantly associated with postoperative depressive symptoms (p = 0.002), whereas age (p = 0.592) and sex (p = 0.069) were not. CONCLUSIONS: Depressive symptoms occur in almost half of stoma surgery patients, which is higher than the general population, and many inflammatory bowel disease and colorectal cancer populations outlined in the literature. However, validated measures suggest this is mostly at a level of clinical severity below major depressive disorder. Stoma patient outcomes and postoperative psychosocial adjustment may be enhanced by increased psychological evaluation and care in the perioperative period.

PROTACs: A Hope for Breast Cancer Patients?

BACKGROUND: Breast Cancer (BC) is the most widely occurring disease in women. A massive number of women are diagnosed with breast cancer, and many lose their lives every year. Cancer is the leading cause of death worldwide, posing a formidable challenge to the current medication difficulties. OBJECTIVES: The main objective of this study is to examine and explore novel therapy (PROTAC) and its effectiveness against breast cancer. METHODS: The literature search was conducted across Medline, Cochrane, ScienceDirect, Wiley Online, Google Scholar, PubMed, and Bentham Sciences from 2001 to 2020. The articles collected were screened, segregated, and selected papers were included for writing the review article. RESULTS AND CONCLUSION: A novel innovation emerged around two decades ago that has great potential to overcome the limitations and provide future direction for the treatment of many diseases, which has presently not many therapeutic options available and are regarded as incurable with traditional techniques. That innovation is called PROTAC (Proteolysis Targeting Chimera), which can efficaciously ubiquitinate and debase cancer, encouraging proteins through noncovalent interaction. PROTACs constituted of two active regions isolated by a linker are equipped for eliminating explicit undesirable protein. It is empowering greater sensitivity to "drugresistant targets" and a more prominent opportunity to influence non-enzymatic function. PROTACs have been demonstrated to show better target selectivity contrasted with traditional small-molecule inhibitors. So far, the most investigation into PROTACs mainly concentrated on cancer treatment applications, including breast cancer. The treatment of different ailments may benefit the patients from this blossoming innovation.

Gastrointestinal recovery after surgery: protocol for a systematic review.

INTRODUCTION: Gastrointestinal recovery after surgery is of worldwide significance. Postoperative gastrointestinal dysfunction is multifaceted and known to represent a major source of postoperative morbidity, however, its significance to postoperative care across all surgical procedures is unknown. The complexity of postoperative gastrointestinal recovery is poorly defined within gastrointestinal surgery, and even less so outside this field. To inform the clinical care of surgical patients worldwide, this systematic review and meta-analysis will aim to characterise the duration of postoperative gastrointestinal recovery that can be expected across all surgical procedures and determine the associations between factors that may affect this. METHODS AND ANALYSIS: MEDLINE, Embase, Cochrane Library and CINAHL will be searched for studies reporting the time to first postoperative passage of stool after any surgical procedure. We will screen records, extract data and assess risk of bias in duplicate. Forest plots will be constructed for time to postoperative gastrointestinal recovery, as assessed by various outcome measures. Because of potential heterogeneity, a random-effects model will be used throughout the meta-analysis. Funnel plots will be used to test for publication bias. Meta-regressions will be undertaken where the outcome is the mean time to first postoperative passage of stool, with potential predictors and confounders being patient characteristics, postoperative outcomes and surgical factors. ETHICS AND DISSEMINATION: This study will not involve human or animal subjects and, thus, does not require ethics approval. The outcomes will be disseminated via publication in peer-reviewed scientific journal(s) and presentations at scientific conferences. PROSPERO REGISTRATION NUMBER: CRD42021256210.

Quality of YouTube Videos on Laparoscopic Cholecystectomy for Patient Education.

BACKGROUND: Surgical patients frequently seek information from digital sources, particularly before common operations such as laparoscopic cholecystectomy (LC). YouTube provides a large amount of free educational content; however, it lacks regulation or peer review. To inform patient education, we evaluated the quality of YouTube videos on LC. METHODS: We searched YouTube with the phrase "laparoscopic cholecystectomy." Two authors independently rated quality of the first 50 videos retrieved using the JAMA, Health on the Net (HON), and DISCERN scoring systems. Data collected for each video included total views, time since upload, video length, total comments, and percentage positivity (proportion of likes relative to total likes plus dislikes). Interobserver reliability was assessed using an intraclass correlation coefficient (ICC). Association between quality and video characteristics was tested. RESULTS: Mean video quality scores were poor, scoring 1.9/4 for JAMA, 2.0/5.0 for DISCERN, and 4.9/8.0 for HON. There was good interobserver reliability with an ICC of 0.78, 0.81, and 0.74, respectively. Median number of views was 21,789 (IQR 3000-61,690). Videos were mostly published by private corporations. No video characteristic demonstrated significant association with video quality. CONCLUSION: YouTube videos for LC are of low quality and insufficient for patient education. Treating surgeons should advise of the website's limitations and direct patients to trusted sources of information.

Separation of sub-micron particles from micron particles using acoustic fluid relocation combined with acoustophoresis.

Acoustophoresis has gained increasing attention as a gentle, non-contact, and high-throughput cell and particle separation technique. It is conveniently used to isolate and enrich particles that are greater than 2 µm; however, its use in manipulating particles smaller than 2 µm is limited. In this work, we present an alternative way of using acoustic forces to manipulate sub-micrometer particles in continuous flow fashion. It has been shown that acoustic forces can be employed to relocate parallel laminar flow streams of two impedance-mismatched fluids. We demonstrate the separation of sub-micron particles from micron particles by the combination of acoustophoresis and acoustic fluid relocation. The micron particles are focused into the middle of the flow channel via primary acoustic forces while sub-micron particles are moved to the side via drag forces created by the relocating fluid. We demonstrate the proof of the concept using binary mixtures of particles comprised of sub-micron/micron particles, micron/micron particles, and bovine red blood cells with E. coli. The efficiency of the particle enrichment is determined via flow cytometry analysis of the collected streams. This study demonstrates that by combining acoustic fluid relocation with acoustophoresis, sub-micron particles can be effectively separated from micron particles at high flow rates and it can be further implemented to separate binary mixtures of micron particles if the volumetric ratio of two particles is greater than 10 and the larger particle diameter is about 10 µm. The combined method is more appropriate to use than acoustophoresis in situations where acoustic streaming and differences in acoustic impedance of fluids can be of concern. Graphical abstract In the presence of a resonance acoustic field, the clean high-density fluid (dark gray) and the low-density sample fluid are relocated. During this process, E. coli are separated from the red blood cells (RBCs).

Simple and inexpensive micromachined aluminum microfluidic devices for acoustic focusing of particles and cells.

We introduce a new method to construct microfluidic devices especially useful for bulk acoustic wave (BAW)-based manipulation of cells and microparticles. To obtain efficient acoustic focusing, BAW devices require materials that have high acoustic impedance mismatch relative to the medium in which the cells/microparticles are suspended and materials with a high-quality factor. To date, silicon and glass have been the materials of choice for BAW-based acoustofluidic channel fabrication. Silicon- and glass-based fabrication is typically performed in clean room facilities, generates hazardous waste, and can take several hours to complete the microfabrication. To address some of the drawbacks in fabricating conventional BAW devices, we explored a new approach by micromachining microfluidic channels in aluminum substrates. Additionally, we demonstrate plasma bonding of poly(dimethylsiloxane) (PDMS) onto micromachined aluminum substrates. Our goal was to achieve an approach that is both low cost and effective in BAW applications. To this end, we micromachined aluminum 6061 plates and enclosed the systems with a thin PDMS cover layer. These aluminum/PDMS hybrid microfluidic devices use inexpensive materials and are simply constructed outside a clean room environment. Moreover, these devices demonstrate effectiveness in BAW applications as demonstrated by efficient acoustic focusing of polystyrene microspheres, bovine red blood cells, and Jurkat cells and the generation of multiple focused streams in flow-through systems. Graphical abstract The aluminum acoustofluidic device and the generation of multinode focusing of particles.

Cone Beam Computed Tomography Study of Root Canal Morphology of Permanent Mandibular Incisors in Indian Subpopulation.

BACKGROUND: The aim of the study was to determine the root canal morphology of permanent mandibular incisor teeth in the Indian subpopulation with the use of cone beam computed tomography (CBCT). MATERIAL/METHODS: CBCT images of 200 patients with 800 permanent mandibular incisors, fulfilling necessary inclusion criteria and aged 18 to 60 years were evaluated. The number of roots, number of root canals and canal configuration were investigated and then classified according to Vertucci's classification of root canals. The effect of gender on the incidence of root canal morphology was also investigated. RESULTS: All the permanent mandibular incisors had a single root. The majority of mandibular incisors (66.5%) had a single root with a single canal. The prevalence of second canals was as follows: right central incisor - 33.5%, left central incisor - 30%, right lateral incisors - 33.5% and left lateral incisor - 36.5%. According to gender, 15.2% of men and 20.4% of women had a second root canal. Type 1 Vertucci configuration was most prevalent, followed by type 3, type 2, type 5 and type 4 in that order. CONCLUSIONS: Type 1 Vertucci's classification (64.5%) was the most prevalent canal configuration in the mandibular anterior teeth in the Indian population. Type 5 Vertucci's classification was the most frequently observed canal configuration of the two-canalled teeth. CBCT is an excellent imaging modality for detection of different canal configurations of mandibular incisors.

A rare cause of perioperative stroke.

Perioperative stroke can occur following 0.2-0.3% of general surgical and orthopedic procedures. We are reporting a patient who developed multiple strokes in the immediate postoperative period following total knee replacement, where etiological workup revealed multiple pulmonary arteriovenous fistulae (PAVF). The significance of PAVF with paradoxical embolism in perioperative settings has rarely been reported in the literature.

Anesthesia for awake craniotomy: a retrospective study.

CONTEXT: Awake craniotomy is increasingly performed the world over. We share our experience of performing craniotomy awake with our anesthetic protocol. AIMS: To evaluate and analyze the anesthesia records of the patients who underwent awake craniotomy at our institution. SETTINGS AND DESIGN: University teaching hospital, Retrospective study. MATERIALS AND METHODS: We reviewed records of the 42 consecutive patients who underwent awake craniotomy under conscious sedation using Fentanyl and Propofol infusion until December 2005. The drugs were titrated (Bispectral monitoring was used in 16 patients) to facilitate intermittent intraoperative neurological testing. All patients received scalp blocks with a mixture of bupivacaine and lignocaine with adrenaline. Haloperidol and ondansetron were administered in all patients at induction of anesthesia. RESULTS: All patients completed the procedure. One patient each needed endotracheal intubation and LMA for airway control during closure, while another required CPAP perioperatively because of desaturation to <80%. There was significantly decreased use of anesthetics (P<0.001) and a trend towards reduction in complications (e.g. respiratory depression and deep sedation) (P>0.05) with the use of BIS as compared to without BIS. Intraoperative complications were hypertension (19%), tight brain (14.2%), focal seizure (9.5%) respiratory depression (7.1%), deep sedation (7.1%), tachycardia (7.1%) and bradycardia. Two patients desaturated to <95%. 23.8% patients developed transient neurological deficits. The most frequent postoperative complications were PONV (19%) and seizures (16.6%). CONCLUSIONS: With the use of advanced monitoring and newer anesthetics, awake craniotomy is a relatively safe procedure with an accepted rate of complications.

Gemcitabine in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy: a phase II California cancer consortium trial.

A phase II trial was designed to evaluate the efficacy and toxicity of gemcitabine in patients with non-small-cell lung cancer (NSCLC) previously treated with platinum-containing regimens and prospectively categorized for platinum response status. Treatment consisted of gemcitabine 1000 mg/m2 given intravenously on days 1 and 8 of a 21-day cycle. The status of p53 in pretreatment tumor tissue was assessed by immunohistochemistry (IHC). Sixty-one patients who progressed or recurred following platinum-based therapy were enrolled, 26 platinum-sensitive and 35 platinum-refractory. A median of 4 treatment courses (range, 2-7 courses) was delivered. Of the 55 patients assessable for response, there was 1 confirmed complete response and 3 with a confirmed partial response for an overall response proportion of 7%. Twenty-one patients had stable disease while 28 progressed and 2 patients had an unconfirmed partial response. Three of the responders (2 confirmed, 1 unconfirmed) were platinum-refractory. Median progression-free survival (PFS) and overall survival for all patients were 4.1 months and 8.6 months, respectively. Median PFS and overall survival for the platinum-sensitive and platinum-refractory cohorts were 5.4 months versus 3.1 months, and 11.9 months versus 7.1 months, respectively. Toxicity was principally hematologic with grade 3/4 neutropenia in 21% and grade 4 platelets in 8%. There were no treatment-related deaths. Twenty-four of 33 patients (73%) had p53-positive tumors. Although no significant association between platinum sensitivity and p53 status was seen, patients with platinum-sensitive disease and negative p53 by IHC had a trend toward longer survival compared to those with platinum-refractory disease and/or p53 positivity (P = 0.06). We concluded that salvage gemcitabine in this dose and schedule is safe and tolerable in previously platinum-treated patients with NSCLC.