CD4+ CAR-T cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR-T cell therapy.

Multiple myeloma is characterized by frequent clinical relapses following conventional therapy. Recently, chimeric antigen receptor T (CAR-T) cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, while >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune intrinsic mechanisms may contribute to this resistance. While there were no pre-existing T cell features associated with clinical outcomes, we found that patients with a durable response to CAR-T cell treatment had greater persistence of their CAR-T cells compared to patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR-T cells. These cells expand in vivo early after infusion but express exhaustion markers (HAVCR2 and TIGIT) and remain polyclonal. Finally, we demonstrate that non-classical monocytes are enriched in the myeloma niche and may induce CAR-T cell dysfunction through mechanisms that include TGFß. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR-T cell therapy.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates.

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there's a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

Immune Activation Profiles Elicited by Distinct, Repeated TLR Agonist Infusions in Rhesus Macaques.

TLR agonists are a promising class of immune system stimulants investigated for immunomodulatory applications in cancer immunotherapy and viral diseases. In this study, we sought to characterize the safety and immune activation achieved by different TLR agonists in rhesus macaques (Macaca mulatta), a useful preclinical model of complex immune interactions. Macaques received one of three TLR agonists, followed by plasma cytokine, immune cell subset representation, and blood cell activation measurements. The TLR4 agonist LPS administered i.v. induced very transient immune activation, including TNF-α expression and monocyte activation. The TLR7/8 agonist 2BXy elicited more persistent cytokine expression, including type I IFN, IL-1RA, and the proinflammatory IL-6, along with T cell and monocyte activation. Delivery of 2BXy i.v. and i.m. achieved comparable immune activation, which increased with escalating dose. Finally, i.v. bacillus Calmette-Guérin (BCG) vaccination (which activates multiple TLRs, especially TLR2/4) elicited the most pronounced and persistent innate and adaptive immune response, including strong induction of IFN-γ, IL-6, and IL-1RA. Strikingly, monocyte, T cell, and NK cell expression of the proliferation marker Ki67 increased dramatically following BCG vaccination. This aligned with a large increase in total and BCG-specific cells measured in the lung. Principal component analysis of the combined cytokine expression and cellular activation responses separated animals by treatment group, indicating distinct immune activation profiles induced by each agent. In sum, we report safe, effective doses and routes of administration for three TLR agonists that exhibit discrete immunomodulatory properties in primates and may be leveraged in future immunotherapeutic strategies.

Safety and feasibility of destination care for bariatric surgery: a single institution retrospective study.

INTRODUCTION: Increasing emphasis on value-based healthcare has prompted both employers and healthcare organizations to develop innovative strategies to supply high quality care to patients. One such strategy is through the bundled care payment model (BCPM). Through this model, our institution partnered with employers from across the country to provide quality care for their members. Patients traveling greater than 2 h driving time from the bariatric center were considered "destination" patients. To properly care for our destination patients, our institution created a "destination bariatric program." We sought to investigate comparative outcomes for the first 100 patients who completed the program. We hypothesized that there would be no difference in patient outcomes or complications between destination and local patient groups undergoing sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB). METHODS AND PROCEDURES: A retrospective cohort analysis of patients undergoing bariatric surgery at a MBSAQIP-accredited bariatric surgery center between May 2019 and October 2021 was conducted. Patients were divided into destination or local patient groups based on participation in the established destination surgery program. Patient demographics, perioperative clinical outcomes, and complications were compared and statistically analyzed using two-sample t-tests, Chi-square tests, Fisher's exact tests, and univariate logistic regressions. RESULTS: This study identified 296 patients, which consisted of destination (n = 110) and local (n = 186) patient cohorts. Patients in the destination group had higher rates of diabetes mellitus (29.1% vs 24.2%, p = 0.029), but otherwise cohorts had similar basic demographics and comorbidities. Outcomes revealed no statistically significant associations between patient cohort (destination versus local) and ED admission (p = 0.305), hospital readmission (p = 0.893), surgical reintervention (p = 0.974), endoscopic-reintervention (p = 0.714), and patient complications in the postoperative period (30 days). CONCLUSION: Participation in destination care programs for bariatric surgery was found to be both safe and feasible. These destination programs represent an opportunity to provide a broader patient population access to complex surgical care.

The impact of interval cholecystectomy timing after percutaneous transhepatic cholecystostomy on post-operative adverse outcomes.

OBJECTIVE: This study aims to explore how timing of interval of cholecystectomy (IC) after percutaneous transhepatic cholecystostomy tube (PTC) placement impacts post-operative outcomes. METHODS: A retrospective database analysis of New York State SPARCs database of IC between 2005 and 2015. The timing for IC ranged between > 1 week and < 2 years. Patients undergoing this procedure were further divided into quartiles using 4-time intervals; 1-5 weeks (Q1), 5-8 weeks (Q2), 8-12 weeks(Q3), and > 12 weeks(Q4). The study's primary outcome was hospital length of stay (LOS). Secondary outcomes included discharge status, 30-day readmission, 30-day ED visit, and 90-day reoperation, surgery type, complication, and bile duct injury. Multivariable regression models were used to compare patients across the four-time intervals after adjusting for confounding factors. RESULTS: A total of 1038 patients with a history of PTC followed by IC between > 1 week and < 2 years were included in the final analysis. The median time to IC was 7.7 weeks. Q2 and Q3 both had a significantly higher median LOS of 3 days versus Q1 and Q4 at median of 5 days (p < 0.0001). Patients from racial and ethnic minorities (e.g., African Americans and Hispanics) were more likely to get their IC after 12 weeks (p < 0.05). Further, Black patients had a significantly higher median LOS than White, non-Hispanic patients (8 days vs 4 days, p < 0.0001) and were more likely to have open procedure. Multivariable regression analysis identified shorter LOS during Q2 (Ratio, 0.76, 95%, 0.67-0.87, p < 0.0001), and Q3 (Ratio 0.75, 95% CI, 065-0.86, p < 0.0001) compared to those who got their IC in Q4. Similar findings exist when comparing Q2 and Q3 to those receiving treatment during Q1. CONCLUSION: A time interval of 5-12 weeks between PTC and IC was associated with a decreased LOS. This study also suggests the persistence of racial disparities among these patients.

Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes.

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.

[S073]-The impact of interval appendectomy timing on follow-up adverse outcomes.

OBJECTIVE: This study aims to compare the timing of interval appendectomy (IA) and its impact on post-operative outcomes. METHODS: A retrospective analysis was performed for adult patients diagnosed with appendicitis between 2006 and 2017. IA was defined as a follow-up appendectomy > 1 week and < 2 years after the initial presentation. Time intervals were divided into 4 groups based on patient quartiles: 1-6 weeks, 7-9 weeks, 10-15 weeks, and > 15 weeks. The primary outcome measure was length of stay (LOS). Secondary outcomes included 30-day readmission and IA post-operative complications. Tertiary outcomes included 30-day mortality and colonoscopy suggesting neoplasm or Inflammatory Bowel Disease. RESULTS: A total of 5069 patients' records whose interval appendectomy fell > 1 week and < 2 years after initial presentation were analyzed. Among them, 1006 (19.85%) underwent an initial percutaneous abscess drainage at diagnosis. The median timing for IA was 9.2 weeks. Patients with IA at 1-6 weeks were more likely to have longer LOS when compared to 7-9 weeks (ratio 1.33, 95% CI 1.2-1.48) and 10-15 weeks (ratio 1.38, 95% CI 1.25-1.52). IA between 7 and 9 weeks (ratio 0.81, 95% CI 0.73-0.89) and 10-15 weeks (ratio 0.78, 95% CI 0.71-0.86) was associated with significantly shorter LOS compared to those receiving the operation after 15 weeks. Further, patients requiring abscess drainage (ratio 1.2, 95% CI 1.13-1.34) or those with comorbidities (ratio 1.51, 95% CI 1.39-1.63) were more likely to have longer LOS at IA. Socioeconomic and demographic differences including Black, Hispanic, and those with Medicare and Medicaid insurance had a greater LOS after their IA. CONCLUSION: LOS remains lowest among patients undergoing IA between 7-9 weeks and 10-15 weeks after initial appendicitis presentation. Patients with lower socioeconomic status or from racial minorities had a longer LOS after IA.

Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce T cell paralysis.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n = 9) and matched healthy donors (n = 8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14CTX can directly suppress CD4+ T cells and induce SOCS3 expression in CD4+ T cells, rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce T cell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance.

Adding Umbralisib and Ublituximab (U2) to Ibrutinib in Patients with CLL: A Phase II Study of an MRD-Driven Approach.

PURPOSE: Ibrutinib has transformed the management of chronic lymphocytic leukemia (CLL), though its use is limited by toxicity and resistance. In this study, we utilized an "add on" approach for patients who had been treated with ibrutinib in the front-line or relapsed/refractory settings with detectable MRD. Umbralisib and ublituximab (U2) were added on to ibrutinib, patients were treated until achieving undetectable-MRD (U-MRD), and then they entered a period of treatment-free observation (TFO). PATIENTS AND METHODS: Patients were eligible if they received ibrutinib in any line of therapy for at least 6 months and had detectable MRD (flow cytometry, <1 cell in 10-4 cutoff for U-MRD). U2 was added to ibrutinib, and patients were monitored serially for MRD. Once U-MRD was achieved or a total of 24 cycles were administered, patients entered a period of TFO. The primary study objective was rate of U-MRD. Secondary endpoints included safety and durability of clinical benefit after treatment discontinuation. RESULTS: Twenty-eight patients were enrolled of whom 27 were evaluable for efficacy. Patients received ibrutinib for a median of 21 months (range 7-67) prior to study enrollment. Fourteen patients (52%) have achieved U-MRD per protocol whereas 78% had at least one U-MRD evaluation. Seventeen patients (63%) have entered TFO after a median of 6.4 months on triplet therapy. Progression-free survival at 12 months was estimated at 95%. Grade ≥3 adverse events were hypertension 7%, diarrhea 4%, and increased ALT/AST 4%. CONCLUSIONS: This triplet approach utilizes the addition of U2 to ibrutinib as an MRD-driven time-limited therapy. This therapy was well tolerated and effective. TFO following this therapy appears durable in ongoing follow-up.

Laparoscopic Heller Myotomy and Dor Fundoplication.

INTRODUCTION: Achalasia is an esophageal motility disorder characterized by disordered esophageal peristalsis with failed relaxation of the lower esophageal sphincter resulting in a functional obstruction.Treatment can include medical, endoscopic, or surgical interventions. Although none of these are curative, they each offer methods to create esophageal outflow. MATERIALS AND METHODS: This article discusses our preferred surgical technique used for laparoscopic Heller myotomy with Dor fundoplication. This technique has been developed over the author's career. CONCLUSION: The technique discussed provides a safe and effective strategy to manage achalasia.

Inhibiting Kiss1 Neurons With Kappa Opioid Receptor Agonists to Treat Polycystic Ovary Syndrome and Vasomotor Symptoms.

CONTEXT: Recent evidence suggests that vasomotor symptoms (VMS) or hot flashes in the postmenopausal reproductive state and polycystic ovary syndrome (PCOS) in the premenopausal reproductive state emanate from the hyperactivity of Kiss1 neurons in the hypothalamic infundibular/arcuate nucleus (KNDy neurons). OBJECTIVE: We demonstrate in 2 murine models simulating menopause and PCOS that a peripherally restricted kappa receptor agonist (PRKA) inhibits hyperactive KNDy neurons (accessible from outside the blood-brain barrier) and impedes their downstream effects. DESIGN: Case/control. SETTING: Academic medical center. PARTICIPANTS: Mice. INTERVENTIONS: Administration of peripherally restricted kappa receptor agonists and frequent blood sampling to determine hormone release and body temperature. MAIN OUTCOME MEASURES: LH pulse parameters and body temperature. RESULTS: First, chronic administration of a PRKA to bilaterally ovariectomized mice with experimentally induced hyperactivity of KNDy neurons reduces the animals' elevated body temperature, mean plasma LH level, and mean peak LH per pulse. Second, chronic administration of a PRKA to a murine model of PCOS, having elevated plasma testosterone levels and irregular ovarian cycles, suppresses circulating levels of LH and testosterone and restores normal ovarian cyclicity. CONCLUSION: The inhibition of kisspeptin neuronal activity by activation of kappa receptors shows promise as a novel therapeutic approach to treat both VMS and PCOS in humans.

Sex-specific pubertal and metabolic regulation of Kiss1 neurons via Nhlh2.

Hypothalamic Kiss1 neurons control gonadotropin-releasing hormone release through the secretion of kisspeptin. Kiss1 neurons serve as a nodal center that conveys essential regulatory cues for the attainment and maintenance of reproductive function. Despite this critical role, the mechanisms that control kisspeptin synthesis and release remain largely unknown. Using Drop-Seq data from the arcuate nucleus of adult mice and in situ hybridization, we identified Nescient Helix-Loop-Helix 2 (Nhlh2), a transcription factor of the basic helix-loop-helix family, to be enriched in Kiss1 neurons. JASPAR analysis revealed several binding sites for NHLH2 in the Kiss1 and Tac2 (neurokinin B) 5' regulatory regions. In vitro luciferase assays evidenced a robust stimulatory action of NHLH2 on human KISS1 and TAC3 promoters. The recruitment of NHLH2 to the KISS1 and TAC3 promoters was further confirmed through chromatin immunoprecipitation. In vivo conditional ablation of Nhlh2 from Kiss1 neurons using Kiss1Cre:Nhlh2fl/fl mice induced a male-specific delay in puberty onset, in line with a decrease in arcuate Kiss1 expression. Females retained normal reproductive function albeit with irregular estrous cycles. Further analysis of male Kiss1Cre:Nhlh2fl/fl mice revealed higher susceptibility to metabolic challenges in the release of luteinizing hormone and impaired response to leptin. Overall, in Kiss1 neurons, Nhlh2 contributes to the metabolic regulation of kisspeptin and NKB synthesis and release, with implications for the timing of puberty onset and regulation of fertility in male mice.

Obstetric and perinatal outcomes for women with pre-existing diabetes in rural compared to metropolitan settings in Victoria, Australia.

BACKGROUND: Pre-existing diabetes in pregnancy is associated with an increased risk of complications. Likewise, living in rural, regional and remote Victoria, Australia, is also associated with poorer health outcomes. There is a gap in the literature with regard to whether Victorian women with pre-existing diabetes experience a greater risk of adverse pregnancy outcomes compared to their metropolitan counterparts. AIM: Our objective is to compare obstetric and perinatal outcomes for women with pre-existing diabetes delivering in rural vs metropolitan hospitals in Victoria, Australia. MATERIALS AND METHODS: Retrospective population-based study using routinely collected state-based data of singleton births to women with type 1 and type 2 diabetes who delivered in metropolitan (n = 3233) and rural hospitals (n = 693) in Victoria, Australia, between 2006-2015. Pearson's χ2 test, Fisher's exact test and MannWhitney U-test were used to compare obstetric and perinatal outcomes between metropolitan and rural locations. RESULTS: Delivery in a rural hospital was associated with higher rates of stillbirth (2.3% vs 1.1%, P = 0.027), macrosomia (25.9% vs 16.9%, P < 0.001), shoulder dystocia (8.4% vs 3.5%, P < 0.001) and admission to the neonatal intensive care unit/special care nursery (73.2% vs 59.3%, P < 0.001). Smoking (18.0% vs 8.9%, P < 0.001), overweight/obesity (P = 0.047) and socioeconomic disadvantage (P < 0.001) were more common in rural women. CONCLUSIONS: Women with pre-existing diabetes who deliver in rural hospitals experience a greater risk of adverse perinatal outcomes and present with increased maternal risk factors. These results suggest a need to improve care for women with pre-existing diabetes in rural Victoria.

A Comparison of Colectomy Outcomes Utilizing Open, Laparoscopic, and Robotic Techniques.

INTRODUCTION: Robotic colectomy could reduce morbidity and postoperative recovery over laparoscopic and open procedures. This comparative review evaluates colectomy outcomes based on surgical approach at a single community institution. METHODS: A retrospective review of all patients who underwent colectomy by a fellowship-trained colon and rectal surgeon at a single institution from 2015 through 2019 was performed, and a cohort developed for each approach (open, laparoscopic, and robotic). 30-day outcomes were evaluated. For dichotomous outcomes, univariate logistic regression models were used to quantify the individual effect of each predictor of interest on the odds of each outcome. Continuous outcomes received a similar approach; however, linear and Poisson regression modeling were used, as appropriate. RESULTS: 115 patients were evaluated: 14% (n = 16) open, 44% (n = 51) laparoscopic, and 42% (n = 48) robotic. Among the cohorts, there was no statistically significant difference in operative time, rate of reoperation, readmission, or major complications. Robotic colectomies resulted in the shortest length of stay (LOS) (Kruskal-Wallis P < .0001) and decreased estimated blood loss (EBL) (Kruskal-Wallis P = .0012). Median age was 63 years (interquartile range [IQR] 53-72). 54% (n = 62) were female. Median American Society of Anesthesiologists physical status classification was 3 (IQR 2-3). Median body mass index was 28.67 (IQR 25.03-33.47). A malignant diagnosis was noted on final pathology in 44% (n = 51). CONCLUSION: Among the 3 approaches, there was no statistically significant difference in 30-day morbidity or mortality. There was a statistically significant decreased LOS and EBL for robotic colectomies.

Differential Gene Expression with an Emphasis on Floral Organ Size Differences in Natural and Synthetic Polyploids of Nicotiana tabacum (Solanaceae).

Floral organ size, especially the size of the corolla, plays an important role in plant reproduction by facilitating pollination efficiency. Previous studies have outlined a hypothesized organ size pathway. However, the expression and function of many of the genes in the pathway have only been investigated in model diploid species; therefore, it is unknown how these genes interact in polyploid species. Although correlations between ploidy and cell size have been shown in many systems, it is unclear whether there is a difference in cell size between naturally occurring and synthetic polyploids. To address these questions comparing floral organ size and cell size across ploidy, we use natural and synthetic polyploids of Nicotiana tabacum (Solanaceae) as well as their known diploid progenitors. We employ a comparative transcriptomics approach to perform analyses of differential gene expression, focusing on candidate genes that may be involved in floral organ size, both across developmental stages and across accessions. We see differential expression of several known floral organ candidate genes including ARF2, BIG BROTHER, and GASA/GAST1. Results from linear models show that ploidy, cell width, and cell number positively influence corolla tube circumference; however, the effect of cell width varies by ploidy, and diploids have a significantly steeper slope than both natural and synthetic polyploids. These results demonstrate that polyploids have wider cells and that polyploidy significantly increases corolla tube circumference.

Community hospital decreases narcotic usage in postoperative bariatric patients.

BACKGROUND: Opioids have long been used as an effective form of analgesia for pain in the postoperative setting; however, their addictive potential and associated complications have become a detriment. There has been an increasing movement to decrease opioid prescribing. OBJECTIVE: The aim of this study was to look at common bariatric surgery procedures at a single institution and compare opioid usage before and after the implementation of a multimodal pain regimen. SETTING: Community program, hospital-employed, and private practice, United States. METHODS: Six hundred twelve laparoscopic gastric bypass and laparoscopic sleeve gastrectomy patients were included in this single-institution retrospective cohort study. Data were obtained from chart review. Comparison was made between patients from 2016 and patients from a 3-month period in 2017 when the new pain management protocol had been instituted. RESULTS: The postoperative opioid usage of 516 patients from 2016 was compared with that of 96 patients from a 3-month period in 2017 after initiating the new pain management protocol. The mean intravenous hydromorphone usage of the control group, 16.0 ± 14.6 morphine milligram equivalent (or 4.0 mg ± .2), over the postoperative inpatient stay decreased to 7.3 ± 6.7 morphine milligram equivalent (or 1.8 mg ± .2) in the study group. This represents a 55% decrease. The study group did show less 30-day postoperative complications compared with the control, 1.04% and 2.13%, respectively, although this was not statistically significant. CONCLUSION: A multimodal pain regimen is an effective way to cut opioid usage with no statistical difference in overall 30-day complications.

Intratumoral CD4+ T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer.

Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8+ T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1.

Early consequences of allopolyploidy alter floral evolution in Nicotiana (Solanaceae).

BACKGROUND: Polyploidy has played a major role in angiosperm evolution. Previous studies have examined polyploid phenotypes in comparison to their extant progenitors, but not in context of predicted progenitor phenotypes at allopolyploid origin. In addition, differences in the trends of polyploid versus diploid evolution have not been investigated. We use ancestral character-state reconstructions to estimate progenitor phenotype at allopolyploid origin to determine patterns of polyploid evolution leading to morphology of the extant species. We also compare trends in diploid versus allopolyploid evolution to determine if polyploidy modifies floral evolutionary patterns. RESULTS: Predicting the ancestral phenotype of a nascent allopolyploid from reconstructions of diploid phenotypes at the time of polyploid formation generates different phenotype predictions than when extant diploid phenotypes are used, the outcome of which can alter conclusions about polyploid evolution; however, most analyses yield the same results. Using ancestral reconstructions of diploid floral phenotypes indicate that young polyploids evolve shorter, wider corolla tubes, but older polyploids and diploids do not show any detectable evolutionary trends. Lability of the traits examined (floral shape, corolla tube length, and corolla tube width) differs across young and older polyploids and diploids. Corolla length is more evolutionarily labile in older polyploids and diploids. Polyploids do not display unique suites of floral characters based on both morphological and color traits, but some suites of characters may be evolving together and seem to have arisen multiple times within Nicotiana, perhaps due to the influence of pollinators. CONCLUSIONS: Young polyploids display different trends in floral evolution (shorter, wider corolla tubes, which may result in more generalist pollination) than older polyploids and diploids, suggesting that patterns of divergence are impacted by the early consequences of allopolyploidy, perhaps arising from genomic shock and/or subsequent genome stabilization associated with diploidization. Convergent evolution in floral morphology and color in Nicotiana can be consistent with pollinator preferences, suggesting that pollinators may have shaped floral evolution in Nicotiana.

Effect of Ovarian Hormones and Mating Experience on the Preference of Female Mice to Investigate Male Urinary Pheromones.

In female mice, the expression of receptive lordosis behavior requires estradiol and progesterone actions in the nervous system; however, the contribution of these hormones to females' motivation to seek out male pheromones is less clear. In an initial experiment, sexually naïve ovary-intact female mice preferred to investigate (make nasal contact with) testes-intact male as opposed to estrous female urine, provided they were in vaginal estrus. In a second experiment, groups of sexually naïve and mating-experienced, ovariectomized females were tested for urinary pheromone preference first without and then with ovarian hormone replacement. Without hormone replacement, sexually naïve ovariectomized females showed no preference for male over female urinary pheromones whereas mating-experienced females preferred to investigate male pheromones. Ovariectomized females in both groups preferred male over female urine after sequential s.c. injections with estradiol benzoate followed 2 days later with progesterone and after prolonged (7 days) exposure to estradiol alone. Our results indicate that in sexually naïve female mice estradiol, perhaps aided by progesterone, is required to motivate a preference to seek out male pheromones whereas after mating experience females' preference to investigate male pheromones persists even in the absence of ovarian hormone action.

DREADD-induced silencing of the medial amygdala reduces the preference for male pheromones and the expression of lordosis in estrous female mice.

Sexually naïve estrous female mice seek out male urinary pheromones; however, they initially display little receptive (lordosis) behavior in response to male mounts. Vomeronasal-accessory olfactory bulb inputs to the medial amygdala (Me) regulate courtship in female rodents. We used a reversible inhibitory chemogenetic technique (Designer Receptors Exclusively Activated by Designer Drugs; DREADDs) to assess the contribution of Me signaling to females' preference for male pheromones and improvement in receptivity normally seen with repeated testing. Sexually naïve females received bilateral Me injections of an adeno-associated virus carrying an inhibitory DREADD. Females were later ovariectomized, treated with ovarian hormones, and given behavioral tests following intraperitoneal injections of saline or clozapine-N-oxide (CNO; which hyperpolarizes infected Me neurons). CNO attenuated females' preference to investigate male vs. female urinary odors. Repeated CNO treatment also slowed the increase in lordosis otherwise seen in females given saline. However, when saline was given to females previously treated with CNO, their lordosis quotients were as high as other females repeatedly given saline. No disruptive behavioral effects of CNO were seen in estrous females lacking DREADD infections of the Me. Finally, CNO attenuated the ability of male pheromones to stimulate Fos expression in the Me of DREADD-infected mice but not in non-infected females. Our results affirm the importance of Me signaling in females' chemosensory preferences and in the acute expression of lordosis. However, they provide no indication that Me signaling is required for the increase in receptivity normally seen after repeated hormone priming and testing with a male.