What Complexity Science Predicts About the Potential of Artificial Intelligence/Machine Learning to Improve Primary Care.

Primary care physicians are likely both excited and apprehensive at the prospects for artificial intelligence (AI) and machine learning (ML). Complexity science may provide insight into which AI/ML applications will most likely affect primary care in the future. AI/ML has successfully diagnosed some diseases from digital images, helped with administrative tasks such as writing notes in the electronic record by converting voice to text, and organized information from multiple sources within a health care system. AI/ML has less successfully recommended treatments for patients with complicated single diseases such as cancer; or improved diagnosing, patient shared decision making, and treating patients with multiple comorbidities and social determinant challenges. AI/ML has magnified disparities in health equity, and almost nothing is known of the effect of AI/ML on primary care physician-patient relationships. An intervention in Victoria, Australia showed promise where an AI/ML tool was used only as an adjunct to complex medical decision making. Putting these findings in a complex adaptive system framework, AI/ML tools will likely work when its tasks are limited in scope, have clean data that are mostly linear and deterministic, and fit well into existing workflows. AI/ML has rarely improved comprehensive care, especially in primary care settings, where data have a significant number of errors and inconsistencies. Primary care should be intimately involved in AI/ML development, and its tools carefully tested before implementation; and unlike electronic health records, not just assumed that AI/ML tools will improve primary care work life, quality, safety, and person-centered clinical decision making.

An immune dysfunction score for stratification of patients with acute infection based on whole-blood gene expression.

Dysregulated host responses to infection can lead to organ dysfunction and sepsis, causing millions of global deaths each year. To alleviate this burden, improved prognostication and biomarkers of response are urgently needed. We investigated the use of whole-blood transcriptomics for stratification of patients with severe infection by integrating data from 3149 samples from patients with sepsis due to community-acquired pneumonia or fecal peritonitis admitted to intensive care and healthy individuals into a gene expression reference map. We used this map to derive a quantitative sepsis response signature (SRSq) score reflective of immune dysfunction and predictive of clinical outcomes, which can be estimated using a 7- or 12-gene signature. Last, we built a machine learning framework, SepstratifieR, to deploy SRSq in adult and pediatric bacterial and viral sepsis, H1N1 influenza, and COVID-19, demonstrating clinically relevant stratification across diseases and revealing some of the physiological alterations linking immune dysregulation to mortality. Our method enables early identification of individuals with dysfunctional immune profiles, bringing us closer to precision medicine in infection.

Clinical nurse specialist prescribing in a cancer centre supportive and palliative care team.

OBJECTIVE: To provide insight into the prescribing practices of three independent nurse prescribers (INPs)/clinical nurse specialists (CNSs) working in a supportive and palliative care team (SPCT) in a district general hospital and a specialist tertiary cancer centre in the UK. METHODS: A prospective review of all consultations and the prescribing activity arising from the consultations (and reasons for non-prescribing following a consultation) of 3 INPs/CNSs between 1 August 2018 and 31 October 2018. RESULTS: Four hundred ninety-three consultations were undertaken on 186 individual patients. Two hundred forty-seven consultations representing 50% resulted in changes to a prescription, and 123 of those consultations representing 50% of prescriptions written resulted in the prescribing of an opioid for moderate-to-severe pain. CONCLUSIONS: This service evaluation demonstrates the potential for INPs in SPCTs to provide comprehensive symptom control while enabling CNSs to practice with a greater degree of autonomy leading to enhanced job satisfaction. On the basis of our experience, we would encourage all palliative care CNSs to undergo training to become INPs (if available).

Targeting ATR as Cancer Therapy: A new era for synthetic lethality and synergistic combinations?

The DNA damage response (DDR) machinery is responsible for detecting DNA damage, pausing the cell cycle and initiating DNA repair. Ataxia telangiectasia and Rad3-related (ATR) protein is a key kinase at the heart of the DDR, responsible for sensing replication stress (RS) and signalling it to S and G2/M checkpoints to facilitate repair. In cancer, loss of G1 checkpoint control and activation of oncogenes that drive replication, result in cancer cells more likely to enter S phase with increased RS. These cancer cells become more reliant on their S and G2/M checkpoints, making this an attractive anti-cancer target. Targeting ATR is the focus of many oncology drug pipelines with a number of potent, selective ATR inhibitors developed, four (M6620, M4344, AZD6738 and BAY1895344) are currently in clinical development. Here we summarise the pre-clinical data supporting the use of ATR inhibitors as monotherapy and in combination with chemotherapy, radiotherapy and novel targeted agents such as PARP inhibitors. We discuss the current clinical trial data and the challenges of taking ATR inhibitors into the clinic and of identifying biomarkers to aid patient selection.

Permanent pacing post-Fontan is not associated with reduced long-term survival.

BACKGROUND: The Fontan procedure is the final stage of surgical palliation for a single-ventricle circulation. Significant complications are common including rhythm disturbance necessitating implantation of a permanent pacemaker. This has been widely considered a negative prognostic indicator. METHODS: This single-centre, retrospective case control study involved all patients who underwent the Fontan procedure at the Leeds Congenital Heart Unit between 1990 and 2015 and have had regular follow-up in Yorkshire and Humber, United Kingdom. 167 Fontan patients were identified of which 2 were excluded for having a pre-procedure pacemaker. Of the remainder, 23 patients required a pacemaker. Outcomes were survival, early and late complications, need for further intervention and oxygen saturation in long-term follow-up. RESULTS: There was no difference in survival (30-day survival pacemaker 92.6%, sinus rhythm 90.5%, p = 0.66, 1-year pacemaker 11.1%, sinus rhythm 10.1%, p = 1). The pacemaker group was more likely to have cerebral or renal complications in the first-year post-procedure (acute kidney injury: sinus rhythm 0.8%, pacemaker 19.1%, p = 0.002). No difference was observed in longer term complications including protein losing enteropathy (sinus rhythm 3.5%, pacemaker 0% p = 1). There was no difference in saturations between the two groups at follow-up. Paced patients were more likely to have required further intervention, with a higher incidence of cardiopulmonary bypass procedures (sinus rhythm 6.3%, pacemaker 35%, p < 0.001). CONCLUSIONS: Despite an increase in early complications and the need for further interventions, pacemaker requirement does not appear to affect long-term survival following the Fontan procedure.

Exploring the Frequency of Homologous Recombination DNA Repair Dysfunction in Multiple Cancer Types.

Dysfunctional homologous recombination DNA repair (HRR), frequently due to BRCA mutations, is a determinant of sensitivity to platinum chemotherapy and poly(ADP-ribose) polymerase inhibitors (PARPi). In cultures of ovarian cancer cells, we have previously shown that HRR function, based upon RAD51 foci quantification, correlated with growth inhibition ex vivo induced by rucaparib (a PARPi) and 12-month survival following platinum chemotherapy. The aim of this study was to determine the feasibility of measuring HRR dysfunction (HRD) in other tumours, in order to estimate the frequency and hence wider potential of PARPi. A total of 24 cultures were established from ascites sampled from 27 patients with colorectal, upper gastrointestinal, pancreatic, hepatobiliary, breast, mesothelioma, and non-epithelial ovarian cancers; 8 were HRD. Cell growth following continuous exposure to 10 µM of rucaparib was lower in HRD cultures compared to HRR-competent (HRC) cultures. Overall survival in the 10 patients who received platinum-based therapy was marginally higher in the 3 with HRD ascites (median overall survival of 17 months, range 10 to 90) compared to the 7 patients with HRC ascites (nine months, range 1 to 55). HRR functional assessment in primary cultures, from several tumour types, revealed that a third are HRD, justifying the further exploration of PARPi therapy in a broader range of tumours.

Glutamatergic Neurokinin 3 Receptor Neurons in the Median Preoptic Nucleus Modulate Heat-Defense Pathways in Female Mice.

We have proposed that arcuate neurons coexpressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons) contribute to hot flushes via projections to neurokinin 3 receptor (NK3R)-expressing neurons in the median preoptic nucleus (MnPO). To characterize the thermoregulatory role of MnPO NK3R neurons in female mice, we ablated these neurons using injections of saporin toxin conjugated to a selective NK3R agonist. Loss of MnPO NK3R neurons increased the core temperature (TCORE) during the light phase, with the frequency distributions indicating a regulated shift in the balance point. The increase in TCORE in the ablated mice occurred despite changes in the ambient temperature and regardless of estrogen status. We next determined whether an acute increase in ambient temperature or higher TCORE would induce Fos in preoptic enhanced green fluorescent protein (EGFP)-immunoreactive neurons in Tacr3-EGFP mice. Fos activation was increased in the MnPO but no induction of Fos was found in NK3R (EGFP-immunoreactive) neurons. Thus, MnPO NK3R neurons are not activated by warm thermosensors in the skin or viscera and are not warm-sensitive neurons. Finally, RNAscope was used to determine whether Tacr3 (NK3R) mRNA was coexpressed with vesicular glutamate transporter 2 or vesicular γ-aminobutyric acid (GABA) transporter mRNA, markers of glutamatergic and GABAergic neurotransmission, respectively. In the MnPO, 94% of NK3R neurons were glutamatergic, but in the adjacent medial preoptic area, 97% of NK3R neurons were GABAergic. Thus, NK3R neurons in the MnPO are glutamatergic and play a role in reducing TCORE but are not activated by warm thermal stimuli (internal or external). These findings suggest that KNDy neurons modulate thermosensory pathways for heat defense indirectly via a subpopulation of glutamatergic MnPO neurons that express NK3R.

Estradiol alters body temperature regulation in the female mouse.

Hot flushes are due to estrogen withdrawal and characterized by the episodic activation of heat dissipation effectors. Recent studies (in humans and rats) have implicated neurokinin 3 (NK3) receptor signaling in the genesis of hot flushes. Although transgenic mice are increasingly used for biomedical research, there is limited information on how 17ß-estradiol and NK3 receptor signaling alters thermoregulation in the mouse. In this study, a method was developed to measure tail skin temperature (TSKIN) using a small data-logger attached to the surface of the tail, which, when combined with a telemetry probe for core temperature (TCORE), allowed us to monitor thermoregulation in freely-moving mice over long durations. We report that estradiol treatment of ovariectomized mice reduced TCORE during the light phase (but not the dark phase) while having no effect on TSKIN or activity. Estradiol also lowered TCORE in mice exposed to ambient temperatures ranging from 20 to 36°C. Unlike previous studies in the rat, estradiol treatment of ovariectomized mice did not reduce TSKIN during the dark phase. Subcutaneous injections of an NK3 receptor agonist (senktide) in ovariectomized mice caused an acute increase in TSKIN and a reduction in TCORE, consistent with the activation of heat dissipation effectors. These changes were reduced by estradiol, suggesting that estradiol lowers the sensitivity of central thermoregulatory pathways to NK3 receptor activation. Overall, we show that estradiol treatment of ovariectomized mice decreases TCORE during the light phase, reduces the thermoregulatory effects of senktide and modulates thermoregulation differently than previously described in the rat.

Evolution through cold and deep waters: the molecular phylogeny of the Lithodidae (Crustacea: Decapoda).

The objectives of this work are to use gene sequence data to assess the hypothesis that the Lithodinae arose from ancestors with uncalcified abdomens in shallow waters of the North-East Pacific, investigate the monophyly and interrelationships of genera within the Lithodinae and to estimate the scale and minimum number of biogeographic transitions from the shallow environment to the deep sea and vice versa. To do this, phylogenetic analysis from three mitochondrial and three nuclear markers was conducted using minimum evolution, maximum likelihood and Bayesian methods. The Lithodinae as defined to include North Pacific genus Cryptolithodes may be paraphyletic, with the Hapalogastrinae and Cryptolithodes as sister taxa. This implies that the soft-bodied abdomen of the Hapalogastrinae might not be plesiomorphic for the Lithodidae. Paralomis, Lopholithodes, Phyllolithodes, Lithodes and Neolithodes share a common ancestor, from which the North Pacific Hapalogastrinae did not descend. Lithodid ancestors are likely to have had a north Pacific, shallow water distribution and to have had planktotrophic larvae. North Pacific genus Paralithodes is paraphyletic; P. brevipes is the most basal member of the genus (as sampled) while P. camtschaticus and P. platypus are more closely related to the genera Lithodes and Neolithodes. Genera Lithodes, Neolithodes and Paralomis (as sampled) are monophyletic if Glyptolithodes is included within Paralomis. Lopholithodes is closely related to, but not included within, the Paralomis genus. Paralomis is divided into at least two major lineages: one containing South Atlantic, West African, and Indian Ocean species, and the other containing Pacific and South American species. Several species of Paralomis do not resolve consistently with any other groups sampled, implying a complex and possibly rapid global evolution early in the history of the genus. Relationships within the Lithodes genus vary between analytical methods, suggesting that conclusions may not be stable. Consistently, however, Indian Ocean and Pacific forms-L. murrayi, L. longispina and L. nintokuae form a group separated from Atlantic species such as L. santolla, L. confundens, L. maja and L. ferox.

Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation.

BACKGROUND AND PURPOSE: The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small-molecule agonist, but the pharmacology of these channels is otherwise limited. EXPERIMENTAL APPROACH: Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca2+ and Tl+ measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta. KEY RESULTS: Modification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 µM Yoda1-induced Ca2+ -entry with IC50 s of 1.3 µM (HEK 293 cells) and 1.5 µM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP-evoked Ca2+ elevation or store-operated Ca2+ entry in HEK 293 cells or Ca2+ entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose-dependent relaxation of aortic rings, which was mediated by an endothelium- and NO-dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1. CONCLUSION AND IMPLICATIONS: Chemical antagonism of Yoda1-evoked Piezo1 channel activity is possible, and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains.

Rural clinical school students do come back: But it may take time

BACKGROUND AND OBJECTIVES: Rural clinical schools (RCSs) help address Australia's rural workforce shortfall, but they require an investment by rural clinicians and communities. Our objective was to determine the location of RCS graduates as one measure of the effectiveness of RCSs. METHOD: This cross-sectional study obtained work location data for Australian National University Medical School (ANUMS) graduates and analysed both RCS and non-RCS data. RESULTS: The percentage of graduates working in rural areas after their fifth postgraduate year (PGY6­11: 34.7%) was significantly greater than that of graduates in PGY1­5 (15.2%, P <0.001). DISCUSSION: Many graduates who trained in rural sites spend time in cities before returning to work in rural areas. This is encouraging for rural clinicians and communities, but it can take time for graduates to return.

Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) follicular signalling is conserved in the mare ovary.

The mare ovary is unique in its anatomical structure; however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and protein localisation of key pathway members (PI3K, AKT1, phosphatase and tensin homologue (PTEN), JAK1, STAT3 and suppressor of cytokine signalling 4 (SOCS4)) was compared in tissue from fetal and adult mare ovaries. Tissue from adult ovaries exhibited significantly increased levels of mRNA expression of PI3K, AKT1, PTEN, JAK1, STAT3 and SOCS4 compared with tissue from fetal ovaries. PI3K, AKT1, JAK1 and STAT3 demonstrated redistributed localisation, from pregranulosa cells in fetal development, to both the oocyte and granulosa cells of follicles in the adult ovary, whilst negative feedback molecules PTEN and SOCS4 were only localised to the granulosa cells in the adult ovary. These findings suggest that the PI3K/AKT and JAK/STAT signalling pathways are utilised during folliculogenesis in the mare, similarly to previously studied mammalian species, and may serve as useful biomarkers for assessment of ovary development in the horse.

Can research that is not intended or unlikely to be published be considered ethical?

BACKGROUND: For research to be ethically acceptable, the potential benefits must justify any risks involved for participants. Dissemination of research findings through publication is one way of creating benefit, but not all researchers intend to publish their research. Other factors, such as lack of size or representativeness, generalisability or innovativeness, or negative findings mean the research is unlikely to be published in a peer-reviewed medical journal. OBJECTIVE: This paper discusses ethical considerations in research where peer-reviewed publication is not intended or unlikely. DISCUSSION: Proposing research that is not intended or unlikely to be published in a peer-reviewed journal does not preclude it from being considered ethical. Additional benefits of such projects may include professional development of investigators, pilot data collection leading to more definitive studies, or developing collaborations with research users that increase relevance and improve utility of findings.

Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa.

Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, low doses of ACR and 4HNE also caused a significant decrease in zona binding. In contrast, exogenous malondialdehyde, a commonly used marker of oxidative stress, had little impact on the various sperm parameters assessed. In accounting for the negative physiological impact of ACR and 4HNE, it was noted that both aldehydes readily adducted to sperm proteins located predominantly within the head, proximal centriole, and tail. The detoxifying activity of mitochondrial aldehyde dehydrogenase 2 appeared responsible for a lack of adduction in the midpiece; however, this activity was overwhelmed by 24 h of electrophilic aldehyde exposure. Sequencing of the dominant proteins targeted for ACR and 4HNE covalent modification identified heat shock protein 90 alpha (cytosolic) class A member 1 and arylsulfatase A, respectively. These collective findings may prove useful in the identification of diagnostic biomarkers of stallion fertility and resolving the mechanistic basis of sperm dysfunction in this species.

Adjusting cryodiluent composition for improved post-thaw quality of rabbit spermatozoa.

Improved fertility following artificial insemination with frozen-thawed spermatozoa would offer rabbit producers faster genetic improvement. Previous work investigating cryoprotectants for rabbit spermatozoa have reported inconsistent results. Semen was collected from three rabbit bucks by artificial vagina and frozen using a standard procedure with varied cryodiluent components. Post-thaw analysis encompassed motility, sperm kinematic parameters and acrosome and membrane integrity. Spermatozoa were evaluated at 0, 2 and 4 h after thawing. Experiment 1 compared diluents with 3.5% dimethyl sulfoxide (DMSO), 1.5% acetamide, 1.75% DMSO + 0.75% acetamide or 3.5% DMSO + 1.5% acetamide. The treatment that resulted in the highest post-thaw motility (P<0.001) and acrosome integrity (P<0.001) was DMSO alone. Experiment 2 compared 3.5, 7 and 10% DMSO in the cryodiluent. The best post-thaw sperm motility (P<0.001) and linearity (P = .002) was in 3.5% DMSO, while 10% DMSO afforded higher acrosome/membrane integrity at this last time point (P<0.05). Experiment 3 varied the cryodiluent to contain either 9 or 17% egg yolk or 9 or 17% low density lipoproteins extracted from whole egg yolk. The treatment with the best post-thaw result was 17% egg yolk (motility, P = 0.01; acrosome/membrane integrity, P<0.001). Experiment 4 compared different carbohydrates in the cryodiluent; 50 mM glucose (TCG), 25 mM glucose with 25 mM sucrose (TCGS low), or 50 mM glucose with 50 mM sucrose (TCGS high). When data were pooled across time points, TCG had significantly higher motility than TCGS high (P = 0.021), but was not different from TCGS low. However, TCG had fewer spermatozoa with intact acrosomes and membranes than both TCGS low and TCGS high (P = .002). Put together, these results indicate that the best cryodiluent for rabbit spermatozoa frozen under the conditions used in this paper is with 7% DMSO and 17% egg yolk in a base medium containing 25 mM glucose and 25 mM sucrose.

Non-surgical sterilisation methods may offer a sustainable solution to feral horse (Equus caballus) overpopulation.

Feral horses are a significant pest species in many parts of the world, contributing to land erosion, weed dispersal and the loss of native flora and fauna. There is an urgent need to modify feral horse management strategies to achieve public acceptance and long-term population control. One way to achieve this is by using non-surgical methods of sterilisation, which are suitable in the context of this mobile and long-lived species. In this review we consider the benefits of implementing novel mechanisms designed to elicit a state of permanent sterility (including redox cycling to generate oxidative stress in the gonad, random peptide phage display to target non-renewable germ cells and the generation of autoantibodies against proteins essential for conception via covalent modification) compared with that of traditional immunocontraceptive approaches. The need for a better understanding of mare folliculogenesis and conception factors, including maternal recognition of pregnancy, is also reviewed because they hold considerable potential in providing a non-surgical mechanism for sterilisation. In conclusion, the authors contend that non-surgical measures that are single shot and irreversible may provide a sustainable and effective strategy for feral horse control.

Impact of interprofessional education about psychological and medical comorbidities on practitioners' knowledge and collaborative practice: mixed method evaluation of a national program.

BACKGROUND: Many patients with chronic physical illnesses have co-morbid psychological illnesses, which may respond to interprofessional collaborative care. Continuing education programs frequently focus on skills and knowledge relevant for individual illnesses, and unidisciplinary care. This study evaluates the impact of "Mind the Gap", an Australian interprofessional continuing education program about management of dual illnesses, on practitioners' knowledge, use of psychological strategies and collaborative practice. METHODS: A 6-h module addressing knowledge and skills needed for patients with physical and psychological co-morbid illnesses was delivered to 837 practitioners from mixed health professional backgrounds, through locally-facilitated workshops at 45 Australian sites. We conducted a mixed-methods evaluation, incorporating observation, surveys and network analysis using data collected, before, immediately after, and three months after training. RESULTS: Six hundred forty-five participants enrolled in the evaluation (58 % GPs, 17 % nurses, 15 % mental health professionals, response rate 76 %). Participants' knowledge and confidence to manage patients with psychological and physical illnesses improved immediately. Among the subset surveyed at three months (response rate 24 %), referral networks had increased across seven disciplines, improvements in confidence and knowledge were sustained, and doctors, but no other disciplines, reported an increase in use of motivational interviewing (85.9 % to 96.8 %) and mindfulness (58.6 % to 74 %). CONCLUSIONS: Interprofessional workshops had an immediate impact on the stated knowledge and confidence of participants to manage patients with physical and psychological comorbidities, which appears to have been sustained. For some attendees, there was a sustained improvement in the size of their referral networks and their use of some psychological strategies.

Aldehyde Dehydrogenase Plays a Pivotal Role in the Maintenance of Stallion Sperm Motility.

Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. Because there is a paucity of studies characterizing the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production and motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1, and ALDH2 isoforms in these cells. Strong correlations (P < 0.001) were found between ALDH expression and various motility parameters of stallion spermatozoa including the percentage of progressive (r = 0.79) and rapidly motile (r = 0.79) spermatozoa, whereas repeated measurements over 24 h revealed highly significant correlations among progressive motility loss, 4HNE accumulation, and ALDH expression (P ≤ 0.001). ALDH inhibition resulted in a spontaneous increase in 4HNE levels in viable cells (21.1 ± 5.8% vs. 42.6 ± 5.2%; P ≤ 0.05) and a corresponding decrease in total motility (41.7 ± 6.2% vs. 6.4 ± 2.6%; P ≤ 0.001) and progressive motility (17.0 ± 4.1% vs. 0.7 ± 0.4%; P ≤ 0.001) of stallion spermatozoa over 24 h. Similarly, inhibition of ALDH in 4HNE-challenged spermatozoa significantly reduced total motility over 4 h (35.4 ± 9.7% vs. 15.3 ± 5.1%, respectively; P ≤ 0.05). This study contributes valuable information about the role of the ALDH enzymes in the maintenance of stallion sperm functionality, with potential diagnostic and in vitro applications for assisted reproductive technologies.

Ablation of KNDy Neurons Results in Hypogonadotropic Hypogonadism and Amplifies the Steroid-Induced LH Surge in Female Rats.

In the human infundibular (arcuate) nucleus, a subpopulation of neurons coexpress kisspeptin and neurokinin B (NKB), 2 peptides required for normal reproductive function. A homologous group of neurons exists in the arcuate nucleus of rodents, termed KNDy neurons based on the coexpression of kisspeptin, NKB, and dynorphin. To study their function, we recently developed a method to selectively ablate KNDy neurons using NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (SAP). Here, we ablated KNDy neurons in female rats to determine whether these neurons are required for estrous cyclicity and the steroid induced LH surge. NK3-SAP or Blank-SAP (control) was microinjected into the arcuate nucleus using stereotaxic surgery. After monitoring vaginal smears for 3-4 weeks, rats were ovariectomized and given 17ß-estradiol and progesterone in a regimen that induced an afternoon LH surge. Rats were killed at the time of peak LH levels, and brains were harvested for NKB and dual labeled GnRH/Fos immunohistochemistry. In ovary-intact rats, ablation of KNDy neurons resulted in hypogonadotropic hypogonadism, characterized by low levels of serum LH, constant diestrus, ovarian atrophy with increased follicular atresia, and uterine atrophy. Surprisingly, the 17ß-estradiol and progesterone-induced LH surge was 3 times higher in KNDy-ablated rats. Despite the marked increase in the magnitude of the LH surge, the number of GnRH or anterior ventral periventricular nucleus neurons expressing Fos was not significantly different between groups. Our studies show that KNDy neurons are essential for tonic levels of serum LH and estrous cyclicity and may play a role in limiting the magnitude of the LH surge.