Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

High-dose intravenous hydromorphone for patients who use opioids in the hospital setting: time to reduce the barriers.

Individuals who use opioids have higher rates of hospitalization compared to the general population. Insufficiently treated withdrawal and pain are major factors contributing to high rates of self-initiated hospital discharges (also referred to as leaving against medical advice) in this population. While injectable opioid agonist therapy is limited or unavailable in the majority of Canadian communities, intravenous hydromorphone (IV HM) is widely available in the hospital setting and high-dose IV HM may be a useful treatment adjunct to improve comfort and engagement in inpatient care for some individuals who use opioids. However, major barriers to its use exist including lack of comfort amongst healthcare providers and hospital policies restricting administration. In this commentary, we highlight the potential usefulness of high-dose IV HM as a treatment adjunct for individuals who use opioids in the hospital setting and advocate for expanded hospital policies to facilitate its use.

A Case Report: Rapid Micro-Induction of Buprenorphine/Naloxone to Administer Buprenorphine Extended-Release in an Adolescent With Severe Opioid Use Disorder.

BACKGROUND AND OBJECTIVES: Buprenorphine extended-release (BUP-XR) is a monthly injectable form of opioid agonist therapy. Before its administration, a minimum 7-day induction period with a transmucosal buprenorphine-containing product is recommended. METHODS: Case report (n = 1). RESULTS: A 16-year-old female with active, severe opioid use disorder (OUD) and stimulant use disorder, hepatitis C virus, co-occurring mental health disorders, and complex social stressors had five recent overdoses requiring naloxone. She had previously been treated with methadone and several trials of sublingual buprenorphine/naloxone, but would quickly discontinue the treatment. Using a rapid micro-induction protocol, buprenorphine/naloxone was administered for 3 days. On day 4, 300 mg BUP-XR was administered subcutaneously. Minimal withdrawal symptoms occurred, despite recent fentanyl use. DISCUSSION AND CONCLUSIONS: A rapid sublingual buprenorphine/naloxone micro-induction was successfully used to initiate BUP-XR, thereby eliminating the abstinence period prior to buprenorphine/naloxone administration, shortening the induction period, and minimizing withdrawal. SCIENTIFIC SIGNIFICANCE: This is the first reported case of using rapid micro-induction as a bridge to initiate BUP-XR. By reducing the length of induction to 4 days and minimizing withdrawal, this induction method can make BUP-XR more accessible to patients who would otherwise refuse the medication due to concerns of enduring withdrawal. (Am J Addict 2020;29:531-535).

CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.

CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa.

Ca2+-binding tyrosine-phosphorylation-regulated protein (CABYR) has been implicated in sperm physiological function in several in vitro studies. It has also been implicated as a potential cause of and diagnostic tool in asthenozoospermic human males. CABYR is known to be localized to the fibrous sheath, an accessory structure in the flagellar principal piece. Utilizing the CRISPR-Cas9 technology, we have knocked out this gene in mice to understand its role in male fertility. Cabyr-knockout male mice showed severe subfertility with a defect in sperm motility as well as a significant disorganization in the fibrous sheath. Further, abnormal configuration of doublet microtubules was observed in the Cabyr-knockout spermatozoa, suggesting that the fibrous sheath is important for the correct organization of the axoneme. Our results show that it is the role of CABYR in the formation of the fibrous sheath that is essential for male fertility.

Postponing Childbearing and Fertility Preservation in Young Professional Women.

OBJECTIVES: To evaluate young women's awareness of ovarian reserve testing and oocyte cryopreservation (OC) and assess how testing ovarian reserve may affect the desire for fertility preservation. METHODS: Three questionnaire-based observational studies were conducted among female students/young professionals 20 years of age and older. The third survey was completed after participants were offered anti-Mullerian hormone (AMH) testing. The main outcomes measured included awareness that OC is available, interest in pursuing fertility preservation, and whether interest would change based on knowledge of declining fertility. RESULTS: The first tier of the study included a survey of a total of 337 women. The majority of female subjects were aware of OC (92.1%). Approximately 38.5% of the women responded that they would consider OC for future fertility purposes. This percentage increased to 60.3% if one was aware her fertility was declining. The second tier of the study included 42 resident/fellow physicians who were offered AMH testing. A survey was completed before and after testing was completed. Approximately 12% of participants stated that their AMH level altered their anticipated age of childbearing, whereas 24% would consider cryopreservation based on their results. The most common concern regarding OC was the cost. CONCLUSIONS: Women should be counseled regarding reproductive aging and options for fertility preservation. Offering ovarian reserve testing and making OC more affordable may increase the number of women who undergo elective OC.

Hepatitis C cascade of care among people who inject drugs in Vancouver, Canada.

BACKGROUND: People who inject drugs (PWID) have high rates of hepatitis C virus (HCV) infection. Little is known about the rates of diagnosis and treatment for HCV among PWID. Therefore, this study aims to characterize the cascade of care in Vancouver, Canada, to improve HCV treatment access and delivery for PWID. METHODS: Data were derived from 3 prospective cohort studies of PWID in Vancouver, Canada, between December 2005 and May 2015. The progression of participants was identified through 5 steps in the cascade of care: (1) chronic HCV; (2) linkage to HCV care; (3) liver disease assessment; (4) initiation of treatment; and (5) completion of treatment. Predictors of undergoing liver disease assessment for HCV treatment were identified using a multivariable extended Cox regression model. RESULTS: Among 1571 participants with chronic HCV, 1359 (86.5%) had ever been linked to care, 1257 (80.0%) had undergone liver disease assessment, 163 (10.4%) had ever started HCV treatment, and 71 (4.5%) had ever completed treatment. In multivariable analyses, human immunodeficiency virus (HIV) seropositivity, use of methadone maintenance therapy, and hospitalization in the past 6 months were independently and positively associated with undergoing liver disease assessment (all P < .001), whereas daily heroin injection was independently and negatively associated with undergoing liver disease assessment (P < .001). CONCLUSIONS: Among this cohort of PWID, few had been started on or completed treatment for HCV. These findings highlight the need to improve the prescribing of HCV treatment among PWID with active substance use.

Small Gold Nanoparticles Interfaced to Electrodes through Molecular Linkers: A Platform to Enhance Electron Transfer and Increase Electrochemically Active Surface Area.

For the smallest nanostructures (<5 nm), small changes in structure can lead to significant changes in properties and reactivity. In the case of nanoparticle (NP)-functionalized electrodes, NP structure and composition, and the nature of the NP-electrode interface have a strong influence upon electrochemical properties that are critical in applications such as amperometric sensing, photocatalysis and electrocatalysis. Existing methods to fabricate NP-functionalized electrodes do not allow for precise control over all these variables, especially the NP-electrode interface, making it difficult to understand and predict how structural changes influence NP activity. We investigated the electrochemical properties of small (dcore < 2.5 nm) gold nanoparticles (AuNPs) on boron doped diamond electrodes using three different electrode fabrication techniques with varying degrees of nanoparticle-electrode interface definition. Two methods to attach AuNPs to the electrode through a covalently bound molecular linker were developed and compared to NP-functionalized electrodes fabricated using solution deposition methods (drop-casting and physiadsorption of a monolayer). In each case, a ferrocene redox probe was tethered to the AuNP surface to evaluate electron transfer through the AuNPs. The AuNPs that were molecularly interfaced with the electrode exhibited nearly ideal, reproducible electrochemical behavior with narrow redox peaks and small peak separations, whereas the solution deposited NPs had broader redox peaks with large peak separations. These data suggest that the molecular tether facilitates AuNP-mediated electron transfer. Interestingly, the molecularly tethered NPs also had significantly more electrochemically active surface area than the solution deposited NPs. The enhanced electrochemical behavior of the molecularly interfaced NPs demonstrates the significant influence of the interface on NP-mediated electron transfer and suggests that similar modified electrodes can serve as versatile platforms for studies and applications of nanoparticles.

CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility.

IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm-egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit. However, the cytoplasmic tail of IZUMO1 in many species is highly variable, ranging from 55 to one amino acid. Therefore, to understand the role of the cytoplasmic tail of IZUMO1 in mouse, we utilised the gene manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression level of IZUMO1, it is dispensable for fertilisation in the mouse.

CRISPR/Cas9-Mediated Rapid Generation of Multiple Mouse Lines Identified Ccdc63 as Essential for Spermiogenesis.

Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.

Nickel-iron oxyhydroxide oxygen-evolution electrocatalysts: the role of intentional and incidental iron incorporation.

Fe plays a critical, but not yet understood, role in enhancing the activity of the Ni-based oxygen evolution reaction (OER) electrocatalysts. We report electrochemical, in situ electrical, photoelectron spectroscopy, and X-ray diffraction measurements on Ni(1-x)Fe(x)(OH)2/Ni(1-x)Fe(x)OOH thin films to investigate the changes in electronic properties, OER activity, and structure as a result of Fe inclusion. We developed a simple method for purification of KOH electrolyte that uses precipitated bulk Ni(OH)2 to absorb Fe impurities. Cyclic voltammetry on rigorously Fe-free Ni(OH)2/NiOOH reveals new Ni redox features and no significant OER current until >400 mV overpotential, different from previous reports which were likely affected by Fe impurities. We show through controlled crystallization that ß-NiOOH is less active for OER than the disordered γ-NiOOH starting material and that previous reports of increased activity for ß-NiOOH are due to incorporation of Fe-impurities during the crystallization process. Through-film in situ conductivity measurements show a >30-fold increase in film conductivity with Fe addition, but this change in conductivity is not sufficient to explain the observed changes in activity. Measurements of activity as a function of film thickness on Au and glassy carbon substrates are consistent with the hypothesis that Fe exerts a partial-charge-transfer activation effect on Ni, similar to that observed for noble-metal electrode surfaces. These results have significant implications for the design and study of Ni(1-x)Fe(x)OOH OER electrocatalysts, which are the fastest measured OER catalysts under basic conditions.

Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes.

The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off-target candidates (exact match 13 nucleotides from 3' target and NGG) found by Bowtie software was 9.2 ± 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes (including two genes previously tested) and obtained mutant mice at a 52.9 ± 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off-target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost-effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.

The evolution of nursing in Australian general practice: a comparative analysis of workforce surveys ten years on.

BACKGROUND: Nursing in Australian general practice has grown rapidly over the last decade in response to government initiatives to strengthen primary care. There are limited data about how this expansion has impacted on the nursing role, scope of practice and workforce characteristics. This study aimed to describe the current demographic and employment characteristics of Australian nurses working in general practice and explore trends in their role over time. METHODS: In the nascence of the expansion of the role of nurses in Australian general practice (2003-2004) a national survey was undertaken to describe nurse demographics, clinical roles and competencies. This survey was repeated in 2009-2010 and comparative analysis of the datasets undertaken to explore workforce changes over time. RESULTS: Two hundred eighty four nurses employed in general practice completed the first survey (2003/04) and 235 completed the second survey (2009/10). Significantly more participants in Study 2 were undertaking follow-up of pathology results, physical assessment and disease specific health education. There was also a statistically significant increase in the participants who felt that further education/training would augment their confidence in all clinical tasks (p < 0.001). Whilst the impact of legal implications as a barrier to the nurses' role in general practice decreased between the two time points, more participants perceived lack of space, job descriptions, confidence to negotiate with general practitioners and personal desire to enhance their role as barriers. Access to education and training as a facilitator to nursing role expansion increased between the two studies. The level of optimism of participants for the future of the nurses' role in general practice was slightly decreased over time. CONCLUSIONS: This study has identified that some of the structural barriers to nursing in Australian general practice have been addressed over time. However, it also identifies continuing barriers that impact practice nurse role development. Understanding and addressing these issues is vital to optimise the effectiveness of the primary care nursing workforce.

Interrater reliability of criterion A of the alternative model for personality disorder (Diagnostic and Statistical Manual for Mental Disorders, Fifth Edition-Section III): A meta-analysis.

The alternative model for personality disorder (AMPD) is currently included in Section III of the Diagnostic and Statistical Manual for Mental Disorders, Fifth Edition (DSM-5). This review sought to summarize the literature concerning the interrater reliability (IRR) of the AMPD. Despite high heterogeneity, meta-analysis provided tentative support for the IRR of Criterion A of the AMPD, with pooled intraclass correlation coefficients (ICCs) for the Level of Personality Functioning Scale (LPFS) and its domains falling above DSM acceptability levels. Subgroup analysis of the LPFS suggested IRR scores could be improved by using a specific AMPD Structured Clinical Interview (SCI). Further research should in particular consider the IRR of Criterion B elements of the AMPD and overall PD diagnosis, where insufficient data were available to draw conclusions in the present study. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Initiations of safer supply hydromorphone increased during the COVID-19 pandemic in Ontario: An interrupted time series analysis.

AIMS: Calls to prescribe safer supply hydromorphone (SSHM) as an alternative to the toxic drug supply increased during the COVID-19 pandemic but it is unknown whether prescribing behaviour was altered. We aimed to evaluate how the number of new SSHM dispensations changed during the pandemic in Ontario. METHODS: We conducted a retrospective interrupted time-series analysis using provincial administrative databases. We counted new SSHM dispensations in successive 28-day periods from March 22, 2016 to August 30, 2021. We used segmented Poisson regression methods to test for both a change in level and trend of new dispensations before and after March 17, 2020, the date Ontario's pandemic-related emergency was declared. We adjusted the models to account for seasonality and assessed for over-dispersion and residual autocorrelation. We used counterfactual analysis methods to estimate the number of new dispensations attributable to the pandemic. RESULTS: We identified 1489 new SSHM dispensations during the study period (434 [mean of 8 per 28-day period] before and 1055 [mean of 56 per 28-day period] during the pandemic). Median age of individuals initiating SSHM was 40 (interquartile interval 33-48) with 61.7% (N = 919) male sex. Before the pandemic, there was a small trend of increased prescribing (incidence rate ratio [IRR] per period 1.002; 95% confidence interval [95CI] 1.001-1.002; p<0.001), with a change in level (immediate increase) at the pandemic date (relative increase in IRR 1.674; 95CI 1.206-2.322; p = 0.002). The trend during the pandemic was not statistically significant (relative increase in IRR 1.000; 95CI 1.000-1.001; p = 0.251). We estimated 511 (95CI 327-695) new dispensations would not have occurred without the pandemic. CONCLUSION: The pandemic led to an abrupt increase in SSHM prescribing in Ontario, although the rate of increase was similar before and during the pandemic. The absolute number of individuals who accessed SSHM remained low throughout the pandemic.

First report of phlebotomine sand flies (Diptera: Psychodidae) in Kansas and Missouri, and a PCR method to distinguish Lutzomyia shannoni from Lutzomyia vexator.

Sand flies Lutzomyia (Psathyromyia) shannoni (Dyar) and Lu. (Helcocyrtomyia) vexator (Coquillet) were collected for the first time in southwest Missouri and southeast Kansas, expanding the known range of these species in North America. Altogether, 680 sand flies (356 males and 324 females) were collected during trapping from May through October 2011 and identified using morphological characters. Of the total sand flies collected, 315 were identified as Lu. shannoni, with 181 individuals (or 26.6% of all sand flies) trapped in Missouri and 134 individuals (or 19.7%) trapped in Kansas. Whereas 358 Lu. vexator were identified from southwest Missouri, only a single specimen was trapped in southeast Kansas. One male Lu. vexator with asymmetric gonostyli was trapped in Missouri. We also developed a polymerase chain reaction protocol to consistently and accurately distinguish Lu. shannoni from Lu. vexator based on presence or absence of a 416 bp fragment from the cytochrome oxidase c subunit 1 gene.

Challenges with buprenorphine inductions in the context of the fentanyl overdose crisis: A case series.

INTRODUCTION AND AIMS: North America is currently experiencing an epidemic of opioid overdose deaths, driven by the proliferation of fentanyl in the street drug market. Although buprenorphine/naloxone (BUP/NX) is an evidence-based, first-line opioid agonist for the management of opioid use disorder, a key challenge in its prescribing lies in the fact that it can precipitate opioid withdrawal during its initial induction process. At this time, there is minimal literature on the BUP/NX induction process in individuals who use illicit fentanyl regularly. DESIGN, METHODS AND RESULTS: A case series from a Vancouver, Canada addiction medicine clinic of three fentanyl-exposed patients who experienced unexpected, precipitated withdrawal when initiating BUP/NX. DISCUSSION AND CONCLUSION: These cases describe incidents of precipitated opioid withdrawal occurring after unusually long periods of fentanyl abstention. Although fentanyl is experienced as a short-acting opioid, the drug persists much longer in the body's peripheral tissues. Here, we highlight the new challenges fentanyl may pose to current BUP/NX induction strategies, and explore the possibility of a long-acting pharmacokinetic effect of fentanyl in the setting of repeated illicit use.

"They have their security, we have our community": Mutual support among people experiencing homelessness in encampments in Toronto during the COVID-19 pandemic.

Unaffordable housing is a growing crisis in Canada, exacerbated by the COVID-19 pandemic, yet perspectives of people living outdoors in encampments have primarily gone unheard. We conducted qualitative interviews with encampment residents to explore how mutual support occurred within the social context of encampments. We found that mutually supportive interactions helped residents meet basic survival needs, as well as health and social needs, and reduced common health and safety risks related to homelessness. The homelessness sector should acknowledge that encampment residents form their own positive communities, and ensure policies and services do not isolate people from these beneficial social connections.

Characterizing safer supply prescribing of immediate release hydromorphone for individuals with opioid use disorder across Ontario, Canada.

BACKGROUND: In response to the ongoing overdose crisis, some clinicians in Canada have started prescribing immediate release hydromorphone (IRH) as an alternative to the toxic unregulated drug supply. This practice is often referred to as safer supply. We aimed to identify and characterize patients receiving safer supply IRH and their prescribers in Ontario. METHODS: Using provincial administrative health data, we identified individuals with opioid use disorder prescribed safer supply IRH from January 2016 to March 2020 and reported the number of initiations over time. We summarized demographic, health, and medication use characteristics among patients who received safer supply IRH, and examined select clinical outcomes including retention and death. Finally, we characterized prescribers of safer supply IRH and compared frequent and infrequent prescribers. RESULTS: We identified 534 initiations of safer supply IRH (447 distinct individuals) from 155 prescribers. Initiations increased over time with a peak in the third quarter of 2019 (103 initiations). Patients' median age was 42 (interquartile range [IQR] 34-50), and most were male (60.2%), urban residents, (96.2%), and in the lowest neighborhood income quintile (55.7%), with 13.9% having overdosed in the previous one year. The prevalence of HIV was 13.9%. The median duration on IRH was 272 days (IQR 30-1,244) and OAT was co-prescribed in 62.9% of courses. Death while receiving IRH or within 7 days of discontinuation was rare (≤5 courses;≤0.94 per person-year for each). CONCLUSIONS: Clinicians are increasingly prescribing safer supply IRH in Ontario. Patients prescribed safer supply IRH had demographic and clinical characteristics associated with high risk of death from opioid-related overdose. Short-term deaths among people receiving safer supply IRH were rare.