Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing.

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation.

Benzofuran Iboga-analogs Modulate Nociception and Inflammation in an Acute Mouse Pain Model.

Pain management following acute injury or post-operative procedures is highly necessary for proper recovery and quality of life. Opioids and non-steroidal anti-inflammatory drugs (NSAIDS) have been used for this purpose, but opioids cause addiction and withdrawal symptoms whereas NSAIDS have several systemic toxicities. Derivatives of the naturally occurring iboga alkaloids have previously shown promising behavior in anti-addiction of morphine by virtue of their interaction with opioid receptors. On this frontier, four benzofuran analogs of the iboga family have been synthesized and their analgesic effects have been studied in formalin induced acute pain model in Swiss albino mice at 30 mg/kg of body weight dose administered intraperitoneally. The antioxidant, anti-inflammatory and neuro-modulatory effects of the analogs were analyzed. Reversal of tail flick latency, restricted locomotion and anxiogenic behavior were observed in iboga alcohol, primary amide and secondary amide. Local neuroinflammatory mediators' substance P, calcitonin gene related peptide, cyclooxygenase-2 and p65 were significantly decreased whereas the depletion of brain derived neurotrophic factor and glia derived neurotrophic factor was overturned on iboga analog treatment. Behavioral patterns after oral administration of the best analog were also analyzed. Taken together, these results show that the iboga family of alkaloid has huge potential in pain management.

Electrochemical NH-Sulfoximidation with α-Keto Acids.

An electrochemical N-acylation of sulfoximine has been achieved via the coupling of α-keto acids and NH-sulfoximines. This process involves the sequential cleavage of C-C bond followed by C(sp2)-N bond formation, with the liberation of H2 and CO2 as the by-products. A library of N-aroylated sulfoximines is produced via the coupling of aroyl and sulfoximidoyl radicals by anodic oxidation under constant current electrolysis (CCE). The compatibility of the present protocol has been demonstrated by coupling of various bio-active compounds, such as NH-sulfoximine derived from (-)-borneol, L-menthol, D-glucose derivative, and some commercial drugs such as flurbiprofen, and ibuprofen. This late-stage functionalization highlights the importance of this sustainable protocol. Besides this, various control experiments and detection of H2 evolution have been performed to support the proposed mechanism.

Synthesis of Self Permeable Antisense PMO Using C5-Guanidino-Functionalized Pyrimidines at the 5'-End Enables Sox2 Downregulation in Triple Negative Breast Cancer Cells.

Delivery of macromolecular drugs inside cells has been a huge challenge in the field of oligonucleotide therapeutics for the past few decades. Earliest natural inspirations included the arginine rich stretch of cell permeable HIV-TAT peptide, which led to the design of several molecular transporters with varying numbers of rigid or flexible guanidinium units with different tethering groups. These transporters have been shown to efficiently deliver phosphorodiamidate morpholino oligonucleotides, which have a neutral backbone and cannot form lipoplexes. In this report, PMO based delivery agents having 3 or 4 guanidinium groups at the C5 position of the nucleobases of cytosine and uracil have been explored, which can be assimilated within the desired stretch of the antisense oligonucleotide. Guanidinium units have been connected by varying the flexibility with either a saturated (propyl) or an unsaturated (propargyl) spacer, which showed different serum dependency along with varied cytoplasmic distribution. The effect of cholesterol conjugation in the delivery agent as well as at the 5'-end of full length PMO in cellular delivery has also been studied. Finally, the efficacy of the delivery has been studied by the PMO mediated downregulation of the stemness marker Sox2 in the triple-negative breast cancer cell line MDA-MB 231. These results have validated the use of this class of delivery agents, which permit at a stretch PMO synthesis where the modified bases can also participate in Watson-Crick-Franklin base pairing for enhanced mRNA binding and protein downregulation and could solve the delivery problem of PMO.

4'-C-Acetamidomethyl-2'-O-methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs.

In this study, we designed the 4'-C-acetamidomethyl-2'-O-methoxyethyl (4'-C-ACM-2'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'-C-ACM-2'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'-C-ACM-2'-O-MOE thymidine modification at the penultimate position of the 3'-end of dT20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'-O-Me, 2'-O-MOE, and 2'-F. In gene silencing studies, we found that the 4'-C-ACM-2'-O-MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'-C-ACM-2'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'-C-ACM-2'-O-MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'-C-ACM-2'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

Girls in scrubs: An ethnographic exploration of the clinical learning environment.

BACKGROUND: Gender bias is an enduring issue in the medical profession despite women being more represented within medical schools and the health care workforce in numerous countries across the world. There have been frequent calls for further exploration of gender-based discriminations within medical education, owing to its lasting impact on student's professional development and career trajectories. This paper presents an ethnographic exploration of the experiences of female medical students and doctors in the clinical learning environment (CLE), aiming to disrupt the cycle of gender inequity in the clinical workplace. METHODS: Our research field involved two teaching wards in a Scottish urban hospital, where 120 h of non-participant observations were conducted over 10 months. Combining purposive and convenience sampling, we conducted 36 individual interviews with key informants, which included medical students, foundation doctors, postgraduate trainees, consultant supervisors, and other health care professionals such as nurses and pharmacists. Data was thematically analysed using Bourdieu's theory of social power reproduction. The research team brought diverse professional backgrounds and perspectives to the exploration of data on gendered encounters. RESULTS: Combining the observational and interview data, five themes were generated, which suggested gender-related differentials in social and cultural capital that the participants acquired in the CLE. Experiences of discriminatory behaviour and stereotypical thought processes impacted the female students' engagement and drive towards learning, implying an adverse influence on habitus. In contrast, the valuable influence of gendered role-models in building confidence and self-efficacy signified a positive transformation of habitus. The research participants displayed considerable internalisation of the gendered processes in the CLE that appeared to be linked to the transient nature of clinical placements. CONCLUSIONS: This research reveals that despite constituting the majority demographic of medical school, female students struggle to gain social and cultural capital. Gendered hierarchies that structure clinical workplaces disadvantage female students and doctors, and the differential experiences transform their habitus. Based on our theoretically informed investigation, we advocate for role-models given their positive impact on students' and doctors' habitus. Additionally, medical educators may consider extended clinical placements that provide opportunities for female students and early-career doctors to secure social and cultural capital through integrating better in health care teams and building meaningful interprofessional relationships.

The Burden and Correlates of Waterpipe (Hookah) Smoking among Adolescents and Youth: A Systematic Review.

BACKGROUND: This systematic review evaluated the available medical literature on the prevalence and trends of waterpipe tobacco smoking among adolescents and youth in jurisdictionally representative populations. METHODS: PubMed, Embase, and Scopus were searched for relevant studies from inception until 31 December 2022 that reported the burden of waterpipe smoking among adolescents and youth (10-24 years of age). We extracted qualitative data on the demographic characteristics, burden, and correlates of waterpipe smoking (PROSPERO ID: CRD42022310982). RESULTS: A total of 2,197 articles were screened and 62 were included in the analysis. The majority (29) of the studies was from the United States of America and there were no studies from the south-east Asian region. The prevalence of ever waterpipe smoking among the 10-24 years age group was noted to be 18.16% (95% CI, 18.03-18.29). The prevalence of current (30-day) waterpipe smoking was 6.43% (95% CI, 6.34-6.50). The age of initiation of waterpipe smoking was variable. The prevalence of waterpipe smoking was higher among males, among those who belong to the high- and middle-income groups, and among university students. The common risk factors of waterpipe smoking included cigarette smoking, alcohol, and substance use. Waterpipe smoking resulted in increased susceptibility to the use of conventional forms of tobacco (e.g. smoking) among those who were never smokers. CONCLUSION: Waterpipe smoking usage was significantly high among adolescents and young adults. Developing regulatory guidelines for water-pipe smoking, surveillance of its use, intervention, and specific policy frameworks may be considered a public health priority.

Synthesis of 5'-Thiol Functionalized Morpholino Oligo-Nucleotide and Subsequent Conjugation with IGT to Improve Delivery and Antisense Efficacy In Vitro.

Thiol functionalized oligonucleotides are useful intermediates for a wide range of applications including DNA nanobiotechnology field through conjugation with various types of probes and cargos. Due to the limitation of synthetic process, phosphorodiamidate morpholino oligonucleotides (PMOs) have not been explored like other oligonucleotides through SH conjugation as mentioned above. In this paper, we report the synthesis of 5'-SH functionalized PMO using a solid support synthesis protocol with an optimized cysteine derived linker so that loading and coupling efficiency of morpholino monomers were effective enough to get a 25-mer 5'-SH functionalized PMO against human Nanog. The PMO with SH functionality was subsequently conjugated with our previously reported Internal Oligo-guanidinium Transporter (IGT) in solution phase to obtain the IGT-PMO conjugate. Interestingly, 5'-conjugated PMO (IGT-PMO) showed 2.5 times better antisense efficacy than 3'-conjugated PMO with IGT (PMO-IGT). 5'-Conjugation enables us to use IGT-PMO for further conjugation at the 3'-N terminal of PMO which was not possible earlier with 5'-OH-PMO-IGT. PMO has become an important class of antisense reagents because four PMO-based drugs have been approved for the treatment of Duchenne muscular dystrophy; hence such an improved result with 5'-modified PMO could be useful for enhancing the therapeutic efficacy of DMD drugs. Similarly, thiol-modified PMO could also be explored like other thiol-containing oligonucleotides for various other applications.

Clinicopathological and Molecular Characteristics of Intraosseous Rhabdomyosarcoma Involving Head and Neck Region: A Systematic Review and Meta-Analysis.

Rhabdomyosarcoma with TFCP2 rearrangement is a newly introduced spindle cell neoplasm showing predilection for craniofacial bones exhibiting highly aggressive nature and poor prognosis. Therefore, an attempt was made to delineate the entity for improved understanding and treatment outcomes through comprehensive analysis of the clinicopathological and molecular characteristics. An electronic search was carried out using MEDLINE by PubMed, Scopus, Google scholar, Cochrane library, and EMBASE databases. Original articles and case reports involving intraosseous rhabdomyosarcoma arising in head and neck region with TFCP2 fusion were included. Data were compiled and risk of bias was analyzed using JBI tool. Thirteen eligible articles were included for the quantitative analysis, which revealed 33 cases with TFCP2 fusion. Majority of the affected individuals were females (58%) with mandible being the common site. Most of the patients died within few months after diagnosis demonstrating a low mean survival rate (30 months). Odds ratio, overall survival and disease-free survival were calculated and analyzed statistically concluding that intraosseous rhabdomyosarcomas harboring TFCP2 fusion are found to be novel and dreadful neoplasms. The predilection for young age with poor prognosis exhibited by these lesions demand early diagnosis and specific treatment planning to curtail mortality.

Nuclear receptor Nr1d1 alleviates asthma by abating GATA3 gene expression and Th2 cell differentiation.

Nuclear receptors are a unique family of transcription factors that play cardinal roles in physiology and plethora of human diseases. The adopted orphan nuclear receptor Nr1d1 is a constitutive transcriptional repressor known to modulate several biological processes. In this study, we found that Nr1d1 plays a decisive role in T helper (Th)-cell polarization and transcriptionally impedes the formation of Th2 cells by directly binding to the promoter region of GATA binding protein 3 (GATA3) gene. Nr1d1 interacts with its cellular companion, the nuclear receptor corepressor and histone deacetylase 3 to form a stable repression complex on the GATA3 promoter. The presence of Nr1d1 also imparts protection against associated inflammatory responses in murine model of asthma and its ligand SR9011 eased disease severity by suppressing Th2 responses. Moreover, Chip-seq profiling uncovered Nr1d1 interactions with other gene subsets that impedes Th2-linked pathways and regulates metabolism, immunity and brain functions, therefore, providing empirical evidence regarding the genetic link between asthma and other comorbid conditions. Thus, Nr1d1 emerges as a molecular switch that could be targeted to subdue asthma.

OpenDeID Pipeline for Unstructured Electronic Health Record Text Notes Based on Rules and Transformers: Deidentification Algorithm Development and Validation Study.

BACKGROUND: Electronic health records (EHRs) in unstructured formats are valuable sources of information for research in both the clinical and biomedical domains. However, before such records can be used for research purposes, sensitive health information (SHI) must be removed in several cases to protect patient privacy. Rule-based and machine learning-based methods have been shown to be effective in deidentification. However, very few studies investigated the combination of transformer-based language models and rules. OBJECTIVE: The objective of this study is to develop a hybrid deidentification pipeline for Australian EHR text notes using rules and transformers. The study also aims to investigate the impact of pretrained word embedding and transformer-based language models. METHODS: In this study, we present a hybrid deidentification pipeline called OpenDeID, which is developed using an Australian multicenter EHR-based corpus called OpenDeID Corpus. The OpenDeID corpus consists of 2100 pathology reports with 38,414 SHI entities from 1833 patients. The OpenDeID pipeline incorporates a hybrid approach of associative rules, supervised deep learning, and pretrained language models. RESULTS: The OpenDeID achieved a best F1-score of 0.9659 by fine-tuning the Discharge Summary BioBERT model and incorporating various preprocessing and postprocessing rules. The OpenDeID pipeline has been deployed at a large tertiary teaching hospital and has processed over 8000 unstructured EHR text notes in real time. CONCLUSIONS: The OpenDeID pipeline is a hybrid deidentification pipeline to deidentify SHI entities in unstructured EHR text notes. The pipeline has been evaluated on a large multicenter corpus. External validation will be undertaken as part of our future work to evaluate the effectiveness of the OpenDeID pipeline.

Placement or displacement: An ethnographic study of space in the clinical learning environment.

PURPOSE: This paper aims to examine the spatial attributes in the hospital ward environment and their impact on medical students' learning and experience of the clinical workplace. MATERIALS AND METHODS: An ethnographic study was conducted in a Scottish teaching hospital, combining observations and interviews over a period of 10 months. Two teaching wards served as the field-sites where approximately 120 h of non-participant observations took place sequentially. In addition, 34 individual interviews were conducted with identified key informants that included medical students, junior doctors, postgraduate trainees, consultant supervisors, ward nurses and hospital pharmacist. A combination of Actor-network Theory (ANT) and Social cognitive theory (SCT) was applied to analyse data pertaining to spatial attributes and their relevance to clinical teaching and learning. RESULTS: Analysis of the observational and interview data led to generation of the following themes: spatial attributes in the clinical workplace can enable or constrain teaching and learning opportunities, inadequate spaces impact students' and junior doctors' sense of value, short clinical rotations influence a sense of ownership of doctors' spaces, and contested nature of space in the clinical environment. Several illustrations of the field-sites help to contextualise the themes and aid in understanding the participants' experiences and perceptions. CONCLUSIONS: Our findings suggest a complex entanglement of space with medical students learning and wellbeing in the clinical workplace. Provision of suitable spaces needs to be a core consideration to realise the full potential of work-based learning in medicine.

Discovery and Validation of Circulating microRNAs as Biomarkers for Epileptogenesis after Experimental Traumatic Brain Injury-The EPITARGET Cohort.

Traumatic brain injury (TBI) causes 10-20% of structural epilepsies and 5% of all epilepsies. The lack of prognostic biomarkers for post-traumatic epilepsy (PTE) is a major obstacle to the development of anti-epileptogenic treatments. Previous studies revealed TBI-induced alterations in blood microRNA (miRNA) levels, and patients with epilepsy exhibit dysregulation of blood miRNAs. We hypothesized that acutely altered plasma miRNAs could serve as prognostic biomarkers for brain damage severity and the development of PTE. To investigate this, epileptogenesis was induced in adult male Sprague Dawley rats by lateral fluid-percussion-induced TBI. Epilepsy was defined as the occurrence of at least one unprovoked seizure during continuous 1-month video-electroencephalography monitoring in the sixth post-TBI month. Cortical pathology was analyzed by magnetic resonance imaging on day 2 (D2), D7, and D21, and by histology 6 months post-TBI. Small RNA sequencing was performed from tail-vein plasma samples on D2 and D9 after TBI (n = 16, 7 with and 9 without epilepsy) or sham operation (n = 4). The most promising miRNA biomarker candidates were validated by droplet digital polymerase chain reaction in a validation cohort of 115 rats (8 naïve, 17 sham, and 90 TBI rats [21 with epilepsy]). These included 7 brain-enriched plasma miRNAs (miR-434-3p, miR-9a-3p, miR-136-3p, miR-323-3p, miR-124-3p, miR-212-3p, and miR-132-3p) that were upregulated on D2 post-TBI (p < 0.001 for all compared with naïve rats). The acute post-TBI plasma miRNA profile did not predict the subsequent development of PTE or PTE severity. Plasma miRNA levels, however, predicted the cortical pathology severity on D2 (Spearman ρ = 0.345-0.582, p < 0.001), D9 (ρ = 0.287-0.522, p < 0.001-0.01), D21 (ρ = 0.269-0.581, p < 0.001-0.05) and at 6 months post-TBI (ρ = 0.230-0.433, p < 0.001-0.05). We found that the levels of 6 of 7 miRNAs also reflected mild brain injury caused by the craniotomy during sham operation (ROC AUC 0.76-0.96, p < 0.001-0.05). In conclusion, our findings revealed that increased levels of neuronally enriched miRNAs in the blood circulation after TBI reflect the extent of cortical injury in the brain but do not predict PTE development.

ScotGEM - an update on Scotland's only graduate-entry rural generalist-focused undergraduate programme, including student career intentions.

INTRODUCTION: In 2016, the Scottish Government commissioned ScotGEM, a generalist focused graduate entry medical programme. The first cohort of 55 students entered in 2018 and will graduate in 2022. Key unique features of ScotGEM include over 50% of clinical education being led by GPs, the creation of a team of dedicated Generalist Clinical Mentors (GCMs) who support this, a geographically dispersed approach to delivery, and a focus on healthcare improvement activities. This presentation will focus on the progress of our inaugural cohort in terms of progression, performance, and career intentions in comparison with the related internationally literature. METHODS: Progression and performance will be reported based upon assessment outcomes. Career intentions were assessed via an electronic questionnaire exploring career preferences, including speciality, location and reasoning distributed to the first three cohorts. We utilised questions derived from key UK and Australian studies to allow direct comparison with the existing literature. RESULTS: The response rate was 77% (126/163). ScotGEM students' progression rate was high and performance directly comparable with Dundee students. A positive attitude towards general practice and emergency medicine careers was reported. A high proportion of students intended to remain in Scotland, with half interested in working in rural or remote settings. DISCUSSION: Overall, results suggest ScotGEM is meeting the aims of its Mission, a finding of key workforce relevance in Scotland and other rural European contexts that supplements the existing international evidence base. The role of GCMs has been instrumental and may be applicable in other areas.

A qualitative perspective on what factors influence ScotGEM students' career intentions.

INTRODUCTION: ScotGEM is a novel graduate medicine programme in Scotland with a rural generalist focus. This survey-based study aimed to assess the career intentions of ScotGEM students and the various factors influencing them. METHODS: An online questionnaire was devised from existing literature that explored students' interest regarding generalist or specialty career, geographical location, and influencing factors. Free-text responses regarding their primary care career interest and their reasoning behind geographical preferences allowed for qualitative content analysis. Responses were coded inductively and categorised into themes by two independent researchers who then compared and finalised the themes. RESULTS: 126/163 (77%) completed the questionnaire. Content analysis of free-text responses in relation to a negative attitude towards a prospective GP career yielded themes: personal aptitude, emotional toll of GP and uncertainty. Themes in relation to desired geographical preference included: family factors, lifestyle issues and perceptions regarding professional and personal development opportunities. DISCUSSION: The qualitative analysis of factors influencing the career intentions of students on the graduate programme is key to understanding what is important to them. Students who have decided against primary care have realised an early aptitude for specialism due to their experiences, while also witnessing the potential emotional toll of primary care. Family needs may already be dictating where they will choose to work in the future. Lifestyle reasons were in favour of both urban and rural careers, with a sizeable number of responses still uncertain. These findings and their implications are discussed in context of existing international literature on rural medical workforce.

Guanidinium-Functionalized Flexible Azaproline Transporter for Efficient Intracellular Delivery of Proapoptotic Peptide and PDL1 Antisense Morpholino Oligo in Human Carcinoma Cells In Vitro.

Cell-penetrating peptides (CPPs) are structurally diverse sophisticated tools endowed with high arginine content, amphipathicity, and well-adopted suitable secondary structures. Despite its capability of breaching the lipid barriers, CPP has major limitations such as in vivo metabolic instability, poor bioavailability, and reduced endosomal escape tendency, which are yet to be improved. In this context, we first have introduced a new class of cellular transporter having a guanidinium-functionalized δ-azaproline (δ-azp)-containing peptide where the δ-azp structurally resembles the "proline" amino acid having an additional "N" at the δ-position. This non-natural peptidic backbone was found to impart proteolytic stability, as reported earlier by our group. Herein, we report the synthesis of a flexible azaproline-tetraguanidinium transporter named FAT along with a revised scalable methodology for δ-azp compared to our previously reported procedure. FAT shows a random-coil-like structure as determined by CD spectroscopy, and is hence structurally different from the polyproline PPII helix. Direct translocation is predicted to be the possible mode of the cellular entrance of FAT into CHO cells when the "Bodipy" fluorophore is covalently attached as the cargo. Simultaneously, two other macromolecular therapeutics, e.g., proapoptotic domain peptide (PAD, a 14-mer peptide) and programmed death ligand 1 (PDL1) morpholino (a 25-mer antisense oligo), were successfully conjugated with FAT and delivered into human carcinoma cells, and their efficacy was analyzed by MTT assay and western blot technique, respectively. Having obtained promising results in internalizing different types of cargos, FAT could be envisaged as a potential drug delivery agent as an alternative to natural CPPs for future application.

Structural Modifications to the Internal Oligoguanidinium Transporter Uncover Two Potent Analogues that Effectively Deliver the Proapoptotic Peptide in Multiple Cancer Cell Lines.

Efficient cytosolic delivery with serum-independent kinetics and low toxicity are the ultimate challenges towards the transformation of an antisense oligonucleotide or a therapeutic peptide to a suitable drug candidate for clinical trials. Most delivery vehicles falter on at least one of the above requirements, which hinders their potential in in vivo models as well. Our previous reports on internal guanidinium transporters (IGTs) have established the diversity of this particular class of molecule with the efficient delivery of antisense phosphorodiamidate morpholino oligonucleotides. In this paper, we report twenty IGTs with different types of evidence-backed structural modifications with different types of head-group linkage R, which significantly change the transfection, toxicity, and endosomal escape. Based on these three criteria, the analogues were sorted systematically to find the more promising IGTs, which were then further examined by LysoTracker studies. Finally, two analogues, with cholesteryl linkage (R = Chol) and pentafluorobenzyl linkage (R = PF Cbz), were selected for a proapoptotic peptide delivery as the final validation using a long-chain di-acid linker conjugation. Detailed mechanistic studies also revealed that the primary pathway of endocytosis is macropinocytosis, and that other pathways play different roles depending on the head group of the IGT. Since endocytosis pathways for entry depend on the nature of the cell line, we have shown the mechanistic variations in two cell lines for validation.

IGT mediated Nanog siRNA delivery in prostate cancer cells improves chemosensitization of Epirubicin in vitro.

Despite the enormous potential of siRNAs to transcriptionally downregulate disease causing proteins in many genetic diseases, efficient delivery and endosomal escape are the two bottlenecks that have resulted in only a handful of FDA approved drugs. In this report, we have successfully delivered siRNA against Nanog with the help of pentafluorobenzyl modified Internal Oligo-guanidinium transporter (IGT) that has previously shown promising results in peptide and antisense morpholino delivery. Nanog downregulation in prostate cancer cell line DU145 in serum containing media led to suppression of associated proteins such as KLF4, FAK and cMyc and also enhanced the chemosensitivity of Epirubicin, an anthracycline based drug, in DU145 cells by associated MDR-1 downregulation in vitro. These results show that IGT is a promising candidate for siRNA delivery and its conjugation with stable siRNAs could enhance the chemotherapeutic efficiency of siRNAs alone and in combination with small molecule-based drugs.

Plasma Neurofilament Light Chain (NF-L) Is a Prognostic Biomarker for Cortical Damage Evolution but Not for Cognitive Impairment or Epileptogenesis Following Experimental TBI.

Plasma neurofilament light chain (NF-L) levels were assessed as a diagnostic biomarker for traumatic brain injury (TBI) and as a prognostic biomarker for somatomotor recovery, cognitive decline, and epileptogenesis. Rats with severe TBI induced by lateral fluid-percussion injury (n = 26, 13 with and 13 without epilepsy) or sham-operation (n = 8) were studied. During a 6-month follow-up, rats underwent magnetic resonance imaging (MRI) (day (D) 2, D7, and D21), composite neuroscore (D2, D6, and D14), Morris-water maze (D35−D39), and a 1-month-long video-electroencephalogram to detect unprovoked seizures during the 6th month. Plasma NF-L levels were assessed using a single-molecule assay at baseline (i.e., naïve animals) and on D2, D9, and D178 after TBI or a sham operation. Plasma NF-L levels were 483-fold higher on D2 (5072.0 ± 2007.0 pg/mL), 89-fold higher on D9 (930.3 ± 306.4 pg/mL), and 3-fold higher on D176 32.2 ± 8.9 pg/mL after TBI compared with baseline (10.5 ± 2.6 pg/mL; all p < 0.001). Plasma NF-L levels distinguished TBI rats from naïve animals at all time-points examined (area under the curve [AUC] 1.0, p < 0.001), and from sham-operated controls on D2 (AUC 1.0, p < 0.001). Plasma NF-L increases on D2 were associated with somatomotor impairment severity (ρ = −0.480, p < 0.05) and the cortical lesion extent in MRI (ρ = 0.401, p < 0.05). Plasma NF-L increases on D2 or D9 were associated with the cortical lesion extent in histologic sections at 6 months post-injury (ρ = 0.437 for D2; ρ = 0.393 for D9, p < 0.05). Plasma NF-L levels, however, did not predict somatomotor recovery, cognitive decline, or epileptogenesis (p > 0.05). Plasma NF-L levels represent a promising noninvasive translational diagnostic biomarker for acute TBI and a prognostic biomarker for post-injury somatomotor impairment and long-term structural brain damage.

Context and mechanisms of interprofessional learning during a Longitudinal Integrated Clerkship.

Longitudinal Integrated Clerkships (LIC) are known to afford several educational advantages to healthcare students including superior team working skills. This paper explores the perceptions of undergraduate medical students who undertook a rural LIC in Scottish primary care setting, to develop an understanding of their interprofessional learning (IPL) during the LIC placement. A qualitative approach was used to explore the lived experience of five LIC alumni who participated in this longitudinal study. They shared their experiences through written and audio diaries over a period of 1-2 months followed by individual semi-structured interviews. Transcripts were thematically analyzed to identify key themes related to IPL during LIC placements. Data from 12 audio and 9 written diaries and 5 interviews generated the following inter-woven themes with regards to various contextual factors, and the prominent generative mechanisms underlying the positive IPL experience: general practice setting afforded interprofessional interactions, longitudinality afforded interprofessional relationships, engagement in nurturing clinical teams, absence of hierarchy, flexibility and autonomy during the LIC, and 'goodwill' expressed toward the LIC programme. The significant interplay of enabling contextual factors and the generative mechanisms operating in the primary care practice environment is presented in context of existing research and proposed future developments.