Frequency of Radiation Therapy-Induced Malignancies in Patients With Li-Fraumeni Syndrome and Early-Stage Breast Cancer and the Influence of Radiation Therapy Technique.

PURPOSE: Breast cancer (BC) is the most common malignancy in female patients with Li-Fraumeni syndrome (LFS), a condition associated with an increased risk of various malignancies, including radiation therapy (RT)-induced malignancies (RIM) within previously irradiated areas. Our study aimed to assess the incidence of RIM in patients with LFS and early-stage BC treated with adjuvant RT, including the effect of RT dose and technique. METHODS AND MATERIALS: We examined patients with a germline pathogenic/likely pathogenic TP53 variant diagnosed with early-stage BC and monitored by a hereditary cancer team at a single cancer center. The study endpoints included RIM frequency, the association of RIM with the dose and type of RT (2-dimensional [2D] RT, 3-dimensional [3D] RT, and intensity modulated RT [IMRT]), and BC recurrence. RESULTS: We analyzed 48 patients with a median age of 39 years (range, 21-62). The majority (71%) had the TP53 R337H variant, and 87% were unaware of their LFS diagnosis at the time of BC treatment. Treatment modalities included mastectomy (62%), (neo)adjuvant chemotherapy (66%), and RT (62%), with RT being more common after breast-conserving surgery (87% vs 46% with mastectomy, P = .010). Among the 30 patients treated with RT, 10% developed RIM in the irradiated field, consisting of 3 soft tissue malignancies. RT dose (≤40.8 or >40.8 Gy) did not influence RIM occurrence, but the type of RT did. RIM was observed in 100% of cases with 2D RT (2/2), 50% with IMRT (1/2), and 0% with 3D RT (0/16) (P = .004). CONCLUSIONS: Our study underscores a concerning rate of RIM after adjuvant RT, emphasizing the importance of a thorough risk-benefit evaluation before recommending RT, with preference for its avoidance if possible. Although subgroup sizes were limited, the risk of RIM appeared to be influenced by the RT technique, with higher rates observed with 2D RT and IMRT compared with 3D RT. Early TP53 testing is essential to guide the BC treatment plan.

The World Health Organization classification of pancreaticobiliary cytopathology stratifies risk of malignancy and outcome for endoscopic ultrasound-guided fine-needle aspiration of the pancreas.

BACKGROUND: The World Health Organization (WHO) has recently published a classification for reporting pancreaticobiliary cytopathology with differences compared to the Papanicolaou Society of Cytopathology (PSC) classification. METHODS: Retrospective data were collected from pancreatic endoscopic ultrasound-guided fine-needle aspirations from 2014 to 2017 at a pancreatic cancer center. Absolute risk of malignancy (AROM), relative risk (to benign), performance characteristics, and overall survival were calculated for the entire cohort with comparison of cysts and solid lesions. RESULTS: In total, 2562 cases were included: 16% cyst (n = 411) and 84% solid (n = 2151). The histologic confirmation rate was 43% (n = 1101) and the median follow-up (for benign) was 56 months. For WHO I-VII, overall AROM (%) was 23, 22, 62, 13, 65, 97, and 100; cyst AROM was 7, 0, 19, 13, 38, 78, and 100; and solid AROM was 50, 29, 70, 15, 100, 99, and 100. For PSC I-VI, overall AROM (%) was 23, 29, 64, 0 (IVa), 60 (IVb), 97, and 100; cyst AROM was 7, 0, 19, 0, 21, 78, and 100; and solid AROM was 50, 35, 73, 0, 92, 99, and 100. The difference in relative risk for a cyst (vs. solid) overall was 0.38 for WHO and 0.26 for PSC. WHO and PSC categories showed stratification for the probability of overall survival. CONCLUSIONS: Cystic versus solid lesion type can dramatically affect AROM, particularly for nondiagnostic (I), benign (II), atypical (III), and WHO V categories. WHO IV conveys a similarly low AROM for cystic and solid types. Both classifications stratify the probability of overall survival, including the newly introduced categories WHO IV and WHO V.

Multidisciplinary Management of Cutaneous Gluteus Vaginal Fistula After Sacrospinous Ligament Fixation.

Introduction: Cutaneous gluteal vaginal fistula is a rare but significant postoperative complication which may present years after sacrospinous ligament fixation (SSLF) surgery There is limited data on the management of cutaneous vaginal fistula following SSLF. Case description: This case report describes a 77-year-old who presents twenty years after SSLF with cutaneous gluteal vaginal abscess and fistula. She underwent successful management with CT-guided percutaneous drainage of gluteal abscess and placement of guiding cutaneous vaginal catheter, laparoscopic pelvic wall dissection and evaluation, and transvaginal localization and removal of the infected permanent suture. Discussion: Multi-disciplinary approach should be considered in the treatment of chronic fistula status post SSLF, including interventional radiology, urogynecology, and minimally invasive gynecologic surgery.

Cytologic features of gynecologic germ cell tumors and carcinomas exhibiting germ cell tumor differentiation.

BACKGROUND: In this study, the authors sought to describe the cytologic features of primary gynecologic germ cell tumors and carcinomas exhibiting germ cell differentiation because little information currently exists. METHODS: An institutional database search was performed to identify histologically confirmed gynecologic germ cell tumors and carcinomas with germ cell tumor differentiation. Available cytologic material was reviewed by three observers, and morphologic features were recorded in addition to patient age at original diagnosis, primary tumor site, site(s) from which the examined cytologic material was obtained, and the type of examined cytologic preparations. RESULTS: In total, 15 cytologic specimens from 12 women (aged 19-82 years) were identified and included touch preparations of core biopsies from various sites (n = 6), fine-needle biopsies (n = 2), pelvic washings (n = 1), ascitic fluids (n = 4), pelvic cyst fluid (n = 1), and endometrial aspirate (n = 1). Of the 12 patients, seven had primary gynecologic germ cell tumors, four had gynecologic (ovarian and endometrial) tumors exhibiting somatic yolk sac tumor-like differentiation, and the remaining patient had an intestinal-type adenocarcinoma arising within an ovarian teratoma. There was morphologic overlap among many of the cases, although cytoplasmic vacuolation/granular cytoplasm was seen in 75% of primary yolk sac tumors or carcinomas with yolk sac tumor differentiation, and dense/squamoid cytoplasm was seen in 100% of teratomatous elements that were sampled. CONCLUSIONS: Germ cell tumors and somatic neoplasms exhibiting germ cell tumor differentiation occurring in adult women share some cytologic features and may be difficult to distinguish from one another, although some tumor types showed characteristic cytomorphologic findings.

Surgical mask wearing behaviour in COVID-19 pandemic and influenza seasons: a cross-sectional study on healthcare professional students and staff's perspective in Southern California.

OBJECTIVE: To investigate healthcare professional staff and students' perception of wearing surgical masks before and after their experience with the COVID-19 pandemic, and to evaluate the impact on mask wearing behaviour in future influenza seasons. DESIGN: Cross-sectional study using anonymous survey. SETTING AND PARTICIPANTS: Healthcare students and staff from a healthcare academic institution in Southern California participated in the mask survey study. Survey results were collected from June to November 2021. A total of 305 respondents responded to the survey, with 173 being healthcare students and 132 being working healthcare staff. OUTCOMES: The study examined respondents' perceptions and hospital mask wearing behaviour before and after their COVID-19 pandemic experience, as well as during previous and future influenza seasons. RESULTS: Two hundred and sixty-four (86.6%) respondents agreed that wearing a surgical mask reduces infection and limits transmission of infectious disease, yet prior to the pandemic, only a small proportion wore a mask in the hospital or during patient care. After experiencing the COVID-19 pandemic, more respondents indicated that they would continue to wear a mask when they are in a hospital in general (n=145, 47.5%), during patient care (n=262, 85.9%), during influenza seasons throughout the hospital (n=205, 67.2%) and during influenza seasons during patient care (n=270, 88.5%). CONCLUSION: The pandemic experience has greatly influenced the health prevention behaviours of healthcare students and staff. After the pandemic, many respondents will continue to practice surgical mask wearing behaviour in the hospital, especially during face-to-face patient care. This demonstrates a significant change in health prevention perceptions among the current and the future generation of healthcare professionals.

The transcriptomic landscape of caudal cell mass in different developmental stages of the chick embryo.

INTRODUCTION: The caudal cell mass (CCM) is an aggregate of undifferentiated pluripotent cells and the main player in secondary neurulation. Previous studies have elucidated the dynamic fate of the multipotent cell lineages, with a recent interest in the neuromesodermal progenitors. However, a transcriptomic analysis of the CCM during secondary neurulation has not been performed yet. METHODS: We analyzed RNA sequencing data of CCM samples at three different developmental stages of chicken embryos; HH16 (largest CCM phase), HH20 (secondary neural tube formation phase), and HH28 (degeneration phase). RESULTS: The transcriptomic profiles were clearly distinguishable according to developmental stage, and HH20 was shown to have not only intermediate, but also unique properties in secondary neurulation. A total of 10,666 differentially expressed genes, including FGF18 and GDF11, were identified and enriched in several gene ontologies related to embryogenesis or organogenesis. We also found that genes encoding transcription factors, such as TWIST2, IRX4, HOXB4, HOXD13, LIN28A, CDX4, and Brachyury, were among the top-ranked differentially expressed genes. CONCLUSION: Through transcriptomic profiling, we provided a picture of the developmental process of the CCM. We identified several key molecules or pathways involved in secondary neurulation and the pathogenesis of related diseases.

Pathogenic variants detected by RNA sequencing in Cornelia de Lange syndrome.

Recent studies suggest that transcript isoforms significantly overlap (approximately 60%) between brain tissue and Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs). Interestingly, 14 cohesion-related genes with variants that cause Cornelia de Lange Syndrome (CdLS) are highly expressed in the brain and LCLs. In this context, we first performed RNA sequencing of LCLs from 22 solved (with pathogenic variants) and 19 unsolved (with no confirmed variants) CdLS cases. Next, an RNA sequencing pipeline was developed using solved cases with two different methods: short variant analysis (for single-nucleotide and indel variants) and aberrant splicing detection analysis. Then, 19 unsolved cases were subsequently applied to our pipeline, and four pathogenic variants in NIPBL (one inframe deletion and three intronic variants) were newly identified. Two of three intronic variants were located at Alu elements in deep-intronic regions, creating cryptic exons. RNA sequencing with LCLs was useful for identifying hidden variants in exome-negative cases.

Prenatal opioid exposure inhibits microglial sculpting of the dopamine system selectively in adolescent male offspring.

The current opioid epidemic has dramatically increased the number of children who are prenatally exposed to opioids, including oxycodone. A number of social and cognitive abnormalities have been documented in these children as they reach young adulthood. However, little is known about the mechanisms underlying developmental effects of prenatal opioid exposure. Microglia, the resident immune cells of the brain, respond to acute opioid exposure in adulthood. Moreover, microglia are known to sculpt neural circuits during typical development. Indeed, we recently found that microglial phagocytosis of dopamine D1 receptors (D1R) in the nucleus accumbens (NAc) is required for the natural developmental decline in NAc-D1R that occurs between adolescence and adulthood in rats. This microglial pruning occurs only in males, and is required for the normal developmental trajectory of social play behavior. However, virtually nothing is known as to whether this developmental program is altered by prenatal exposure to opioids. Here, we show in rats that maternal oxycodone self-administration during pregnancy leads to reduced adolescent microglial phagocytosis of D1R and subsequently higher D1R density within the NAc in adult male, but not female, offspring. Finally, we show prenatal and adult behavioral deficits in opioid-exposed offspring, including impaired extinction of oxycodone-conditioned place preference in males. This work demonstrates for the first time that microglia play a key role in translating prenatal opioid exposure to changes in neural systems and behavior.

Relapse-like behavior and nAChR sensitization following intermittent access nicotine self-administration.

Many tobacco smokers consume nicotine intermittently, but the underlying mechanisms and neurobiological changes associated with intermittent nicotine intake are unclear. Understanding intermittent nicotine intake is a high priority, as it could promote therapeutic strategies to attenuate tobacco consumption. We examined nicotine intake behavior and neurobiological changes in male rats that were trained to self-administer nicotine during brief (5 min) trials interspersed with longer (15 min) drug-free periods. Rats readily adapted to intermittent access (IntA) SA following acquisition on a continuous access (ContA) schedule. Probabilistic analysis of IntA nicotine SA suggested reduced nicotine loading behavior compared to ContA, and nicotine pharmacokinetic modeling revealed that rats taking nicotine intermittently may have increased intake to maintain blood levels of nicotine that are comparable to ContA SA. After IntA nicotine SA, rats exhibited an increase in unreinforced responses for nicotine-associated cues (incubation of craving) and specific alterations in the striatal proteome after 7 days without nicotine. IntA nicotine SA also induced nAChR functional upregulation in the interpeduncular nucleus (IPN), and it enhanced nicotine binding in the brain as determined via [11C]nicotine positron emission tomography. Reducing the saliency of the cue conditions during the 5 min access periods attenuated nicotine intake, but incubation of craving was preserved. Together, these results indicate that IntA conditions promote nicotine SA and nicotine seeking after a nicotine-free period.

Differential behavioral functioning in the offspring of rats with high vs. low self-administration of the opioid agonist remifentanil.

Opioid use disorder (OUD) has a variety of adverse effects on both the users and their offspring. In the current study, a random group of Sprague-Dawley rats (25 females and 15 males) were tested for intravenous self-administration of the opioid agonist remifentanil to determine the range of acquisition for opioid. One-month after the end of self-administration of remifentanil, rats with the highest intake were mated together and rats with lowest intake were mated together. Then, the offspring of the two groups were tested for anxiety-like behavior, locomotor activity, nociception and intravenous remifentanil self-administration. The parents showed a range of remifentanil self-administration, especially in the female rats. The offspring of the parents with low and high remifentanil self-administration showed significant differences in specific behavioral functions. On the hotplate test of nociception, the female offspring parents with high remifentanil self-administration had significantly longer hotplate latencies, indicating reduced nociception, than the female offspring of parents with low remifentanil-self-administration, whereas there was no difference in the male offspring of low and high responding parents. In the elevated plus maze test of anxiety-like behavior, the offspring of the parents with high remifentanil intake showed more anxiety-like behavior than the offspring of the parents with low remifentanil intake regardless of sex. Locomotor activity was not significantly different. Interestingly, no significant differences in remifentanil self-administration in the offspring of parents with low and high remifentanil self-administration were detected. Overall, our data suggest a considerable range in remifentanil self-administration in rats and the offspring of rats with high opioid self-administration exhibit different behaviors vs offspring of rats with low opioid self-administration.

Spatiotemporal contribution of neuromesodermal progenitor-derived neural cells in the elongation of developing mouse spinal cord.

AIMS: During vertebrate development, the posterior end of the embryo progressively elongates in a head-to-tail direction to form the body plan. Recent lineage tracing experiments revealed that bi-potent progenitors, called neuromesodermal progenitors (NMPs), produce caudal neural and mesodermal tissues during axial elongation. However, their precise location and contribution to spinal cord development remain elusive. MAIN METHODS: Here we used NMP-specific markers (Sox2 and BraT) and a genetic lineage tracing system to localize NMP progeny in vivo. KEY FINDINGS: Sox2 and BraT double positive cells were initially located at the tail tip, but were later found in the caudal neural tube, which is a unique feature of mouse development. In the neural tube, they produced neural progenitors (NPCs) and contributed to the spinal cord gradually along the AP axis during axial elongation. Interestingly, NMP-derived NPCs preferentially contributed to the ventral side first and later to the dorsal side at the lumbar spinal cord level, which may be associated with atypical junctional neurulation in mice. SIGNIFICANCE: Our current observations detail the contribution of NMP progeny to spinal cord elongation and provide insights into how different species uniquely execute caudal morphogenesis.

Penta-fluorophenol: a Smiles rearrangement-inspired cysteine-selective fluorescent probe for imaging of human glioblastoma.

Two of the most critical factors for the survival of glioblastoma (GBM) patients are precision diagnosis and the tracking of treatment progress. At the moment, various sophisticated and specific diagnostic procedures are being used, but there are relatively few simple diagnosis methods. This work introduces a sensing probe based on a turn-on type fluorescence response that can measure the cysteine (Cys) level, which is recognized as a new biomarker of GBM, in human-derived cells and within on-site human clinical biopsy samples. The Cys-initiated chemical reactions of the probe cause a significant fluorescence response with high selectivity, high sensitivity, a fast response time, and a two-photon excitable excitation pathway, which allows the imaging of GBM in both mouse models and human tissue samples. The probe can distinguish the GBM cells and disease sites in clinical samples from individual patients. Besides, the probe has no short or long-term toxicity and immune response. The present findings hold promise for application of the probe to a relatively simple and straightforward following of GBM at clinical sites.

Differential Nicotinic Modulation of Glutamatergic and GABAergic VTA Microcircuits.

Ventral tegmental area (VTA) neurons receive glutamatergic and/or GABAergic input from other local neurons within the VTA. Nicotinic acetylcholine receptor (nAChR) activity is capable of modulating such intra-VTA transmission, but the mechanisms are unclear. Here, we isolated monosynaptic glutamate or GABA transmission from mouse medial VTA (mVTA) to lateral VTA (latVTA) using pharmacology and optogenetics, and we studied the ability of nicotine to modulate these modes of transmission. The action of nicotine on mVTA to latVTA glutamate transmission was bidirectional; nicotine enhanced glutamate release in half of the recorded latVTA cells and inhibited release in the other half. Nicotine-mediated reduction in glutamate release was reversed by blockade of GABAA receptors. This, coupled with expression data demonstrating coexpression of vesicular glutamate transporter 2 (VGluT2) and glutamate decarboxylase 2 (Gad2) in mVTA neurons, suggests that nicotine is able to stimulate GABA corelease from mVTA VGluT2+ neurons. Nicotine had an altogether different effect on mVTA to latVTA GABA release from Gad2+ cells; nicotine suppressed GABA release from mVTA Gad2+ terminals in nearly all cells tested. Together, these data uncover a complex system of local circuitry in the VTA that is modulated by nAChR activity. These actions of nicotine, which occurred at concentrations of nicotine found in the artificial CSF of cigarette smokers, may play a role in the adaptive response of the reward system to repeated nicotine exposure.

Comprehensive genetic analysis of 57 families with clinically suspected Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder with specific dysmorphic features. Pathogenic genetic variants encoding cohesion complex subunits and interacting proteins (e.g., NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major causes of CdLS. However, there are many clinically diagnosed cases of CdLS without pathogenic variants in these genes. To identify further genetic causes of CdLS, we performed whole-exome sequencing in 57 CdLS families, systematically evaluating both single nucleotides variants (SNVs) and copy number variations (CNVs). We identified pathogenic genetic changes in 36 out of 57 (63.2 %) families, including 32 SNVs and four CNVs. Two known CdLS genes, NIPBL and SMC1A, were mutated in 23 and two cases, respectively. Among the remaining 32 individuals, four genes (ANKRD11, EP300, KMT2A, and SETD5) each harbored a pathogenic variant in a single individual. These variants are known to be involved in CdLS-like. Furthermore, pathogenic CNVs were detected in NIPBL, MED13L, and EHMT1, along with pathogenic SNVs in ZMYND11, MED13L, and PHIP. These three latter genes were involved in diseases other than CdLS and CdLS-like. Systematic clinical evaluation of all patients using a recently proposed clinical scoring system showed that ZMYND11, MED13L, and PHIP abnormality may cause CdLS or CdLS-like.

Chronic Nicotine Exposure Alters the Neurophysiology of Habenulo-Interpeduncular Circuitry.

Antagonism of nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb) or interpeduncular nucleus (IPN) triggers withdrawal-like behaviors in mice chronically exposed to nicotine, implying that nicotine dependence involves the sensitization of nicotinic signaling. Identification of receptor and/or neurophysiological mechanisms underlying this sensitization is important, as it could promote novel therapeutic strategies to reduce tobacco use. Using an approach involving photoactivatable nicotine, we previously demonstrated that chronic nicotine (cNIC) potently enhances nAChR function in dendrites of MHb neurons. However, whether cNIC modulates downstream components of the habenulo-interpeduncular (Hb-IP) circuit is unknown. In this study, cNIC-mediated changes to Hb-IP nAChR function were examined in mouse (male and female) brain slices using molecular, electrophysiological, and optical techniques. cNIC enhanced action potential firing and modified spike waveform characteristics in MHb neurons. Nicotine uncaging revealed nAChR functional enhancement by cNIC on proximal axonal membranes. Similarly, nAChR-driven glutamate release from MHb axons was enhanced by cNIC. In IPN, the target structure of MHb axons, neuronal morphology, and nAChR expression is complex, with stronger nAChR function in the rostral subnucleus [rostral IPN (IPR)]. As in MHb, cNIC induced strong upregulation of nAChR function in IPN neurons. This, coupled with cNIC-enhanced nicotine-stimulated glutamate release, was associated with stronger depolarization responses to brief (1 ms) nicotine uncaging adjacent to IPR neurons. Together, these results indicate that chronic exposure to nicotine dramatically alters nicotinic cholinergic signaling and cell excitability in Hb-IP circuits, a key pathway involved in nicotine dependence.SIGNIFICANCE STATEMENT This study uncovers several neuropharmacological alterations following chronic exposure to nicotine in a key brain circuit involved in nicotine dependence. These results suggest that smokers or regular users of electronic nicotine delivery systems (i.e., "e-cigarettes") likely undergo sensitization of cholinergic circuitry in the Hb-IP system. Reducing the activity of Hb-IP nAChRs, either volitionally during smoking cessation or inadvertently via receptor desensitization during nicotine intake, may be a key trigger of withdrawal in nicotine dependence. Escalation of nicotine intake in smokers, or tolerance, may involve stimulation of these sensitized cholinergic pathways. Smoking cessation therapeutics are only marginally effective, and by identifying cellular/receptor mechanisms of nicotine dependence, our results take a step toward improved therapeutic approaches for this disorder.

Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission.

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse ß2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.

Nicotinic Cholinergic Receptors in VTA Glutamate Neurons Modulate Excitatory Transmission.

Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

Photoactivatable drugs for nicotinic optopharmacology.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Food restriction-induced changes in motivation differ with stages of the estrous cycle and are closely linked to RFamide-related peptide-3 but not kisspeptin in Syrian hamsters.

We tested the hypothesis that the effects of food restriction on behavioral motivation are mediated by one or both of the RFamide peptides, RFamide-related peptide-3 (RFRP-3) and kisspeptin (Kp) in female Syrian hamsters (Mesocricetus auratus). Female hamsters fed ad libitum and given a choice between food and adult male hamsters are highly motivated to visit males instead of food on all four days of the estrous cycle, but after 8days of mild food restriction (75% of ad libitum intake) they shift their preference toward food every day of the estrous cycle until the day of estrus, when they shift their preference back toward the males. In support of a role for RFRP-3 in these behavioral changes, the preference for food and the activation of RFRP-3-immunoreactive (Ir) cells in the dorsomedial hypothalamus (DMH) showed the same estrous cycle pattern in food-restricted females, but no association was observed between behavior and the activation of Kp cells in the hypothalamic arcuate nucleus or preoptic area. Next, we tested the hypothesis that food-restriction-induced activation of RFRP-3-Ir cells is modulated by high levels of ovarian steroids at the time of estrus. In support of this idea, on nonestrous days, mild food restriction increased activation of RFRP-3-Ir cells, but failed to do so on the day of estrus even though this level of food restriction did not significantly decrease circulating concentrations of estradiol or progesterone. Furthermore, in ovariectomized females, food-restriction-induced increases in activation of RFRP-3-Ir cells were blocked by systemic treatment with progesterone alone, estradiol plus progesterone, but not estradiol alone. Central infusion with RFRP-3 in ad libitum-fed females significantly decreased sexual motivation and produced significant increases in 90-minute food hoarding, in support of the hypothesis that elevated central levels of RFRP-3 are sufficient to create the shift in behavioral motivation in females fed ad libitum. Together, these results are consistent with the hypothesis that high levels of ingestive motivation are promoted during the nonfertile phase of the estrous cycle by elevated activation of RFRP-3-Ir cells, and RFRP-3-Ir cellular activation is modulated by ovarian steroids around the time of estrus, thereby diverting attention away from food and increasing sexual motivation.

Mutant Cas9-transcriptional activator activates HIV-1 in U1 cells in the presence and absence of LTR-specific guide RNAs.

CRISPR/Cas9 systems have been advanced as promising tools in the HIV eradication armamentarium for sequence-specific disruption or latency reversal. Enthusiasm is balanced by concerns about off-target host genome modification and effects on HIV evolution. In the chronically HIV-1-infected U1 promonocytic latency model, we have confirmed stimulation of HIV-1 production by a mutant Cas9-transcriptional activator and guide RNAs with two guide RNAs apparently more potent than one. However, significant increases were also observed in the absence of guide RNAs. We encourage continued careful evaluation of non-sequence-specific and off-target effects of Cas9-mediated approaches.