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Importance: Stereotactic body radiotherapy (SBRT) is widely used for stage I medically inoperable non-small cell lung cancer (NSCLC), yet varied results from randomized clinical trials (RCTs) and concerns in treating centrally located tumors persist. Objective: To examine whether SBRT would improve local control (LC) compared with hypofractionated conventional radiotherapy (CRT). Design, Setting, and Participants: This phase 3 RCT was conducted in 16 Canadian centers. Patients with medically inoperable stage I (≤5 cm) NSCLC were randomized 2:1 to SBRT of 48 Gy in 4 fractions (peripheral NSCLC) or 60 Gy in 8 fractions (central NSCLC) vs CRT of 60 Gy in 15 fractions. Data were collected from May 2014 to January 2020, and data were analyzed from July 2022 to July 2023. Interventions: SBRT or CRT. Main Outcomes and Measures: The primary objective was to determine the effectiveness of SBRT compared with CRT based on LC at 3 years. Secondary outcomes included event-free survival, overall survival, and toxic effects. All radiation plans were subject to real-time/final review. Local failures were centrally adjudicated. The study was designed to detect a 3-year LC improvement of SBRT from 75% to 87.5%. The target sample size was 324 patients. Results: Of 233 included patients, 119 (51.1%) were male, and the mean (SD) age was 75.4 (7.7) years; the median (IQR) follow-up was 36.1 (26.4-52.8) months. A total of 154 patients received SBRT and 79 received CRT. The 3-year LC was 87.6% (95% CI, 81.9%-93.4%) for SBRT and 81.2% (95% CI, 71.9%-90.5%) for CRT (hazard ratio [HR], 0.61; 95% CI, 0.31-1.20; P = .15). The HR was 1.02 (95% CI, 0.72-1.45; P = .87) for event-free survival and 1.18 (95% CI, 0.80-1.76; P = .40) for overall survival. Minimal acute toxic effects were observed. Among those randomized to SBRT, late grade 3 or 4 toxic effects occurred in 5 of 45 (11%) with central NSCLC and 2 of 109 (1.8%) with peripheral NSCLC; among those randomized to CRT, in 1 of 19 (5%) with central NSCLC and 1 of 60 (2%) with peripheral NSCLC. One patient who received SBRT for an ultracentral lesion (target overlapping proximal bronchus) experienced a possible treatment-related grade 5 event (hemoptysis). Conclusions and Relevance: This RCT compared lung SBRT with hypofractionated CRT that included central/ultracentral tumors. No difference was detected in LC between groups. Severe toxic effects were limited, including patients with central tumors. The trial provides important prospective data evaluating SBRT; however, further research is necessary to determine if SBRT is more effective than CRT for peripheral and central NSCLC. Trial Registration: ClinicalTrials.gov Identifier: NCT03924869.
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BACKGROUND: To examine sex differences in overdose (OD) mortality based upon substances involved. METHODS: A retrospective database analysis of West Virginia OD decedents (12,666 unintentional OD deaths, 2005-early 2023). Exposures were substances judged to contribute to death. The main outcome measure was determination of male to female death ratios with varying co-intoxicant involvement, particularly related to alcohol and fentanyl. Secondary outcomes included associations of fentanyl concentrations with alcohol concentrations and male sex, including fentanyl (F) and inactive metabolite norfentanyl (N) concentration variability between sexes. RESULTS: Alcohol co-intoxication in OD deaths was associated with higher male:female death ratios, from 2.0 (alcohol absent) to 3.3 (alcohol present). There was a greater increase over time in alcohol involvement in recent deaths involving females compared to males (relative increases of 52% vs. 6%, respectively). Male:female ratios with alcohol and fentanyl co-involvement ranged from 5.9:1 (only two drugs involved) to 2.4:1 (= 5 substances), with females significantly more likely to have multiple substances contributing to death. Overall, males had statistically significantly larger fentanyl (F) to norfentanyl (N) median concentration ratios compared to females (8.8 vs. 6.9, respectively). Multivariable analyses found alcohol presence was associated with a statistically significant 22% reduction in predicted fentanyl concentrations. CONCLUSIONS: Male:female ratios in unintentional OD deaths were higher with greater alcohol involvement and lower with fewer co-intoxicants. Fentanyl and norfentanyl concentration differences by sex were observed. It is important to determine possible contributors to sex differences in OD death rates to better target prevention and treatment initiatives.
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Probiotic research has undergone some exciting and unanticipated changes in direction since the 2010 commentary by GSH, which speculated on probiotics being ultimately utilized as "factories" capable of releasing pharmaceutical-grade metabolites with therapeutic potential for a wide range of primarily gastrointestinal disorders. Indeed, the unrelenting search for new alternatives to antibiotics has further stimulated the development of "next-generation" probiotics. Postbiotics, defined as inanimate microorganisms and/or their components that confer a health benefit on the host, remain at the forefront of current probiotic research, with increasing numbers of probiotic species, strains, and substrains now being identified and further exploited as pharmabiotics; probiotics with a proven pharmacologic role in health and disease that have been subjected to clinical trial prior to approval by regulatory bodies. However, perhaps the most unanticipated probiotic development over the past 15 y has been the emergence of psychobiotics with the potential to improve aspects of mental health, such as depression and anxiety, through the release of bioactive metabolites. Moreover, the recent identification of pharmacobiotics, probiotics capable of facilitating the effectiveness of conventional pharmaceutical drugs, is opening new avenues for probiotic applications to combat a range of diseases, including cancers of the digestive system. Although in its infancy, recent reports of oncobiotics with antineoplastic properties are further expanding the potential for certain next-generation probiotics to impact current cancer treatment regimens and possibly even contribute to cancer prevention. Looking to the next 15 y of probiotic development, one could perhaps predict the ultimate development of regulatory-approved xenopostbiotic formulations comprising metabolites with the capacity to improve digestive health, decrease the severity of intestinal disease, and increase the effectiveness of conventional pharmaceuticals, whereas simultaneously improving cognitive functioning and mental welfare. Although speculative, these xenopostbiotic formulations could prove especially effective for the adjunctive treatment of serious chronic diseases such as cancer.
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Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer survival but are associated with treatment-limiting hypertension, often attributed to endothelial cell (EC) dysfunction. Using phosphoproteomic profiling of VEGFRi-treated ECs, drugs were screened for mitigators of VEGFRi-induced EC dysfunction and validated in primary aortic ECs, mice, and canine cancer patients. VEGFRi treatment significantly raised systolic blood pressure (SBP) and increased markers of endothelial and renal dysfunction in mice and canine cancer patients. α-Adrenergic-antagonists were identified as drugs that most oppose the VEGFRi proteomic signature. Doxazosin, one such α-antagonist, prevented EC dysfunction in murine, canine, and human aortic ECs. In mice with sorafenib-induced-hypertension, doxazosin mitigated EC dysfunction but not hypertension or glomerular endotheliosis, while lisinopril mitigated hypertension and glomerular endotheliosis without impacting EC function. Hence, reversing EC dysfunction was insufficient to mitigate VEGFRi-induced-hypertension in this mouse model. Canine cancer patients with VEGFRi-induced-hypertension were randomized to doxazosin or lisinopril and both agents significantly decreased SBP. The canine clinical trial supports safety and efficacy of doxazosin and lisinopril as antihypertensives for VEGFRi-induced-hypertension and the potential of trials in canines with spontaneous cancer to accelerate translation. The overall findings demonstrate the utility of phosphoproteomics to identify EC-protective agents to mitigate cardio-oncology side effects.
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Doxazossina , Células Endoteliais , Hipertensão , Receptores de Fatores de Crescimento do Endotélio Vascular , Animais , Cães , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Doxazossina/farmacologia , Doxazossina/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteômica/métodos , Pressão Sanguínea/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Lisinopril/farmacologia , Lisinopril/uso terapêutico , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 1/uso terapêutico , Sorafenibe/farmacologia , Sorafenibe/uso terapêuticoRESUMO
Diving marine mammals are a diverse group of semi- to completely aquatic species. Some species are targets of conservation and rehabilitation efforts; other populations are permanently housed under human care and may contribute to clinical and biomedical investigations. Veterinary medical care for species under human care, at times, may necessitate the use of general anesthesia for diagnostic and surgical indications. However, the unique physiologic and anatomic adaptations of one representative diving marine mammal, the bottlenose dolphin, present several challenges in providing ventilatory and cardiovascular support to maintain adequate organ perfusion under general anesthesia. The goal of this review is to highlight the unique cardiopulmonary adaptations of the completely aquatic bottlenose dolphin (Tursiops truncatus), and to identify knowledge gaps in our understanding of how those adaptations influence their physiology and pose potential challenges for sedation and anesthesia of these mammals.
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Adaptação Fisiológica , Golfinho Nariz-de-Garrafa , Mergulho , Animais , Golfinho Nariz-de-Garrafa/fisiologia , Mergulho/fisiologia , AnestesiaRESUMO
Selective serotonin reuptake inhibitors (SSRIs) including citalopram are commonly used antidepressants that can be involved in drug-related deaths along with opioids and other substances. This study characterized citalopram involvement in West Virginia (WV) drug-related deaths compared to other SSRI and non-SSRI-related deaths. All 2005-2021 WV drug-related deaths were analyzed in this retrospective study. Demographics, other substances involved, and comorbidities in cases in which citalopram was listed on the death certificate were compared to other SSRI-related and total non-SSRI deaths. Citalopram concentrations and the association between citalopram presence with predicted fentanyl concentrations were determined. Citalopram was the most common antidepressant present in the deaths (4.5% of 14,363 total), with most (81%) unintentional. Male: female ratios in citalopram cases (0.9:1) were significantly lower than in non-SSRI deaths (2.3:1). Almost two-thirds of citalopram deaths had ≥ 4 substances involved compared to 26% of non-SSRI deaths. Overall, oxycodone was most frequently identified in citalopram deaths (fentanyl more commonly in recent years), followed by alprazolam and diazepam. Cardiovascular comorbidity was significantly more common in citalopram than non-SSRI deaths. No association was found between citalopram presence and predicted fentanyl concentrations. Most citalopram-related deaths were unintentional and involved proportionately more females, with larger numbers of concurrent substances present and more cardiovascular comorbidity compared to non-SSRI deaths. Citalopram is widely used and less toxic than many antidepressants. The extent to which it contributed to overdose deaths can be difficult to ascertain given the multiple substances usually present.
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Background: Toxoplasma gondii is a pathogenic parasite with worldwide distribution. We investigated curcumin and chitosan in combination on the viability of T. gondii tachyzoites in silico, in vitro and in vivo. Methods: A 3D model was employed in Urmia University of Medical Sciences, Urmia, Iran in 2021 to study the interaction between curcumin and dihydrofolate reductase (DHFR). Ramachandran root-mean-square deviation and VERIFY3D validated the model. Cytotoxicity of curcumin and chitosan was evaluated by MTT viability assay. BALB/c mice infected with 104 Toxoplasma organisms were treated with curcumin, chitosan, and the combination of curcumin+chitosan. Serum levels of inducible NO synthetase (iNOs), interferon gamma (IFN-γ), interleukin (IL)-5, glutamate oxaloacetic transaminases(SGOT), and glutamic pyruvate transaminase (SGPT) were determined. Result: Curcumin-DHFR and curcumin-DHPS (dihydropteroate synthase) interactions and calculated enzyme energy indicated an excellent affinity for curcumin with DHFR, but not DHPS. MTT results of concurrent treatments demonstrated IC50 rates of 0.1, 0.05, and 0.01 mg/ml at 24, 48, and 72h, respectively. IFN-γ, IL-5 and iNOs levels in curcumin+chitosan treated mice were 1.71, 0.51, and 1.51 IU/L, while those of SGOT and SGPT were 76 and 84 IU/L, respectively. Conclusion: The combination of curcumin and chitosan increased survival time of infected mice by seven days. Curcumin and chitosan in combination regulated the immune system and reduced liver damage, potentially forming the basis of a new treatment for toxoplasmosis.
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BACKGROUND: Pneumococcal carriage in children has been extensively studied, but carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is less understood. METHODS: Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by PCR. Pneumococcal carriage in adults 18-44 years was compared with carriage among PCV-vaccinated children 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and IPD data for England for the same ages for 2014-2019. Age-group specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for adults aged 65+ years. RESULTS: In total 98 isolates (97 carriers) were identified from 1,631 adults aged 18+ years (age and sex standardized carriage prevalence 6.4%), with only three identified solely by PCR. Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R=0.9). Serotypes 3, 37 and 8 represented a higher proportion of adult carriage than expected from direct low-level transmission from children to adults. The predicted carriage serotype distributions for 65+ years aligned more closely with the carriage serotype distribution for young adults than young children. CONCLUSIONS: The nasal wash technique is highly sensitive; additional benefit of PCR is limited. Comparison of carriage serotype distributions suggests some serotypes may be circulating preferentially within these specific young adults. Our data suggest that for some serotypes carried by adults 65+ years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered.
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The dystrophin protein has well-characterized roles in force transmission and maintaining membrane integrity during muscle contraction. Studies have reported decreased expression of dystrophin in atrophying muscles during wasting conditions, and that restoration of dystrophin can attenuate atrophy, suggesting a role in maintaining muscle mass. Phosphorylation of S3059 within the cysteine-rich region of dystrophin enhances binding between dystrophin and ß-dystroglycan, and mimicking phosphorylation at this site by site-directed mutagenesis attenuates myotube atrophy in vitro. To determine whether dystrophin phosphorylation can attenuate muscle wasting in vivo, CRISPR-Cas9 was used to generate mice with whole body mutations of S3059 to either alanine (DmdS3059A) or glutamate (DmdS3059E), to mimic a loss of, or constitutive phosphorylation of S3059, on all endogenous dystrophin isoforms, respectively. Sciatic nerve transection was performed on these mice to determine whether phosphorylation of dystrophin S3059 could attenuate denervation atrophy. At 14 days post denervation, atrophy of tibialis anterior (TA) but not gastrocnemius or soleus muscles, was partially attenuated in DmdS3059E mice relative to WT mice. Attenuation of atrophy was associated with increased expression of ß-dystroglycan in TA muscles of DmdS3059E mice. Dystrophin S3059 phosphorylation can partially attenuate denervation-induced atrophy, but may have more significant impact in less severe modes of muscle wasting.
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Distrofina , Músculo Esquelético , Atrofia Muscular , Animais , Fosforilação , Camundongos , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Atrofia Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Distrofina/metabolismo , Distrofina/genética , Masculino , Denervação Muscular/métodos , Camundongos Endogâmicos C57BLRESUMO
Background: Adverse events in early life can have impact lasting into adulthood. We investigated the long-term effects of systemic inflammation during postnatal development on adult microglial responses to LPS in two CNS regions (cortex, cervical spinal cord) in male and female rats. Methods: Inflammation was induced in Sprague-Dawley rats by lipopolysaccharide (LPS, 1 mg/kg) administered intraperitoneally during postnatal development at P7, P12 or P18. As adults (12 weeks of age), the rats received a second LPS dose (1 mg/kg). Control rats received saline. Microglia were isolated 3 hours post-LPS from the cortex and cervical spinal cord. Gene expression was assessed via qRT-PCR for pro-inflammatory (IL-6, iNOS, Ptgs2, C/EBPb, CD14, CXCL10), anti-inflammatory (CD68, Arg-1), and homeostatic genes (P2Y12, Tmemm119). CSF-1 and CX3CL1 mRNA was analyzed in microglia-free homogenates. Results: Basal gene expression in adult microglia was largely unaffected by early life LPS. Changes in adult microglial pro-inflammatory genes in response to LPS were either unchanged or attenuated in rats exposed to LPS during postnatal development. Ptgs2, C/EBPb, CXCL10 and Arg-1 were the genes most affected, with expression levels significantly downregulated vs control rats without postnatal LPS exposure. Cortical microglia were affected more by postnatal inflammation than spinal microglia, and males were more impacted than females. Overall, inflammatory challenge at P18 had the greatest effect on adult microglial gene expression, whereas challenge at P7 had less impact. Microglial homeostatic genes were unaffected by postnatal LPS. Conclusions: Long-lasting effects of postnatal inflammation on adult microglia depend on the timing of postnatal inflammation, CNS region and sex.
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INTRODUCTION: Soft tissue sarcomas (STSs) are rare and diverse primary malignant tumors that comprise approximately 1% of all malignancies. Misdiagnoses and unplanned excisions of STSs are common due to the tumor's rarity, leading to secondary tumor bed excisions (TBEs). Reconstructive outcomes for TBEs remain poorly understood, prompting this study to address the knowledge gap and inform preoperative discussions. METHODS: This was a retrospective cohort study of patients who underwent STS excisions at a quaternary cancer center. Patients were categorized into mass excision (ME) and TBE groups. Reconstructive approaches were divided into simple (primary closure, complex repair, skin grafts, local flaps) and advanced (pedicled or free flaps). The groups were compared for postoperative outcomes, including complications, recurrence, and death. RESULTS: When simple reconstructive techniques were used, TBEs exhibited higher rates of overall and major complications, whereas MEs had higher rates of overall and minor complications. Intergroup analysis revealed that with simple reconstruction, rates of overall and major complications were higher in TBEs than in MEs, and rates of minor complications were higher in MEs than in TBEs. Regression analyses revealed that simple reconstruction of TBEs had 90% and 180% higher odds of major complications and reoperation compared to simple reconstruction of MEs ( P < 0.05). CONCLUSION: TBEs, despite their smaller size, exhibited a heightened susceptibility to overall and major complications, challenging the notion that simpler techniques suffice in these cases. Our findings encourage the consideration of advanced reconstructive techniques for TBEs that may seem amenable to simple reconstructive techniques.
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Procedimentos de Cirurgia Plástica , Complicações Pós-Operatórias , Sarcoma , Humanos , Sarcoma/cirurgia , Estudos Retrospectivos , Feminino , Masculino , Procedimentos de Cirurgia Plástica/métodos , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Idoso , Adulto , Retalhos Cirúrgicos/transplante , Neoplasias de Tecidos Moles/cirurgia , Estudos de Coortes , Resultado do Tratamento , Reoperação/estatística & dados numéricosRESUMO
Excessive accumulation of intermuscular adipose tissue (IMAT) is a common pathological feature in various metabolic and health conditions and can cause muscle atrophy, reduced function, inflammation, insulin resistance, cardiovascular issues, and unhealthy aging. Although IMAT results from fat accumulation in muscle, the mechanisms underlying its onset, development, cellular components, and functions remain unclear. IMAT levels are influenced by several factors, such as changes in the tissue environment, muscle type and origin, extent and duration of trauma, and persistent activation of fibro-adipogenic progenitors (FAPs). FAPs are a diverse and transcriptionally heterogeneous population of stromal cells essential for tissue maintenance, neuromuscular stability, and tissue regeneration. However, in cases of chronic inflammation and pathological conditions, FAPs expand and differentiate into adipocytes, resulting in the development of abnormal and ectopic IMAT. This review discusses the role of FAPs in adipogenesis and how they remodel IMAT. It highlights evidence supporting FAPs and FAP-derived adipocytes as constituents of IMAT, emphasizing their significance in adipose tissue maintenance and development, as well as their involvement in metabolic disorders, chronic pathologies and diseases. We also investigated the intricate molecular pathways and cell interactions governing FAP behavior, adipogenesis, and IMAT accumulation in chronic diseases and muscle deconditioning. Finally, we hypothesize that impaired cellular metabolic flexibility in dysfunctional muscles impacts FAPs, leading to IMAT. A deeper understanding of the biology of IMAT accumulation and the mechanisms regulating FAP behavior and fate are essential for the development of new therapeutic strategies for several debilitating conditions.
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Adipogenia , Tecido Adiposo , Humanos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Células-Tronco/metabolismo , Células-Tronco/citologia , Adipócitos/metabolismo , Adipócitos/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/citologia , Diferenciação CelularRESUMO
Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude once respiratory neural activity is restored. Such long-lasting facilitation represents a form of respiratory motor plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although facilitation also occurs in inspiratory intercostal nerve activity after diminished respiratory neural activity (iIMF), it is of shorter duration. Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and specific isoform of the relevant atypical PKC are unknown. Here, we used RNA interference to test the hypothesis that the zeta atypical PKC isoform (PKCζ) within phrenic motor neurons is necessary for iPMF but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural injections of siRNAs targeting PKCζ (siPKCζ) to knock down PKCζ mRNA within phrenic and intercostal motor neurons were made in rats. Control rats received a nontargeting siRNA (NTsi) or an active siRNA pool targeting a novel PKC isoform, PKCθ (siPKCθ), which is required for other forms of respiratory motor plasticity. Phrenic nerve burst amplitude and external intercostal (T2) electromyographic (EMG) activity were measured in anesthetized and mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e., neural apnea) created by modest hypocapnia (20 min) or a similar recording duration without neural apnea (time control). Phrenic burst amplitude was increased in rats treated with NTsi (68 ± 10% baseline) and siPKCθ (57 ± 8% baseline) 60 min after neural apnea vs. time control rats (-3 ± 3% baseline), demonstrating iPMF. In contrast, intrapleural siPKCζ virtually abolished iPMF (5 ± 4% baseline). iIMF was transient in all groups exposed to neural apnea; however, intrapleural siPKCζ attenuated iIMF 5 min after neural apnea (50 ± 21% baseline) vs. NTsi (97 ± 22% baseline) and siPKCθ (103 ± 20% baseline). Neural inactivity elevated the phrenic, but not intercostal, responses to hypercapnia, an effect that was blocked by siPKCζ. We conclude that PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient iIMF.NEW & NOTEWORTHY We report important new findings concerning the mechanisms regulating a form of spinal neuroplasticity elicited by prolonged inhibition of respiratory neural activity, inactivity-induced phrenic motor facilitation (iPMF). We demonstrate that the atypical PKC isoform PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient inspiratory intercostal facilitation. Our findings are novel and advance our understanding of mechanisms contributing to phrenic motor plasticity.
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Neurônios Motores , Nervo Frênico , Proteína Quinase C , Ratos Sprague-Dawley , Animais , Nervo Frênico/fisiologia , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Neurônios Motores/fisiologia , Masculino , Ratos , Plasticidade Neuronal/fisiologiaRESUMO
Microglia are innate CNS immune cells that play key roles in supporting key CNS functions including brain plasticity. We now report a previously unknown role for microglia in regulating neuroplasticity within spinal phrenic motor neurons, the neurons driving diaphragm contractions and breathing. We demonstrate that microglia regulate phrenic long-term facilitation (pLTF), a form of respiratory memory lasting hours after repetitive exposures to brief periods of low oxygen (acute intermittent hypoxia; AIH) via neuronal/microglial fractalkine signaling. AIH-induced pLTF is regulated by the balance between competing intracellular signaling cascades initiated by serotonin vs adenosine, respectively. Although brainstem raphe neurons release the relevant serotonin, the cellular source of adenosine is unknown. We tested a model in which hypoxia initiates fractalkine signaling between phrenic motor neurons and nearby microglia that triggers extracellular adenosine accumulation. With moderate AIH, phrenic motor neuron adenosine 2A receptor activation undermines serotonin-dominant pLTF; in contrast, severe AIH drives pLTF by a unique, adenosine-dominant mechanism. Phrenic motor neuron fractalkine knockdown, cervical spinal fractalkine receptor inhibition on nearby microglia, and microglial depletion enhance serotonin-dominant pLTF with moderate AIH but suppress adenosine-dominant pLTF with severe AIH. Thus, microglia play novel functions in the healthy spinal cord, regulating hypoxia-induced neuroplasticity within the motor neurons responsible for breathing.
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A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H-13C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the ß-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.
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INTRODUCTION: A diagnosis of oral squamous cell carcinoma in adolescent patients is extremely rare. When an oral squamous cell carcinoma lesion arises near the teeth and/or periodontium, it can be easily misdiagnosed as an inflammatory condition of endodontic or periodontal origin. METHODS: This is a case report of an otherwise healthy 14-year-old patient who was referred for endodontic evaluation and treatment of a soft-tissue swelling in the anterior maxilla. RESULTS: The unexpected definitive diagnosis of invasive oral squamous cell carcinoma underscores the importance of proper diagnostic testing. CONCLUSIONS: Accurate interpretation of pulp testing results, periapical and cone beam computed tomography imaging, timely biopsy, and prompt definitive treatment are critical when a lesion of nonodontogenic origin is suspected.
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Low-dose (< 2 h/day), acute intermittent hypoxia (AIH) elicits multiple forms of serotonin-dependent phrenic motor plasticity and is emerging as a promising therapeutic strategy to restore respiratory and non-respiratory motor function after spinal cord injury (SCI). In contrast, high-dose (> 8 h/day), chronic intermittent hypoxia (CIH) undermines some forms of serotonin-dependent phrenic motor plasticity and elicits pathology. CIH is a hallmark of sleep disordered breathing, which is highly prevalent in individuals with cervical SCI. Interestingly, AIH and CIH preconditioning differentially impact phrenic motor plasticity. Although mechanisms of AIH-induced plasticity in the phrenic motor system are well-described in naïve rats, we know little concerning how these mechanisms are affected by chronic SCI or intermittent hypoxia preconditioning. Thus, in a rat model of chronic, incomplete cervical SCI (lateral spinal hemisection at C2 (C2Hx), we assessed serotonin type 2A, 2B and 7 receptor expression in and near phrenic motor neurons and compared: 1) intact vs. chronically injured rats; and 2) the impact of preconditioning with varied "doses" of intermittent hypoxia (IH). While there were no effects of chronic injury or intermittent hypoxia alone, CIH affected multiple receptors in rats with chronic C2Hx. Specifically, CIH preconditioning (8 h/day; 28 days) increased serotonin 2A and 7 receptor expression exclusively in rats with chronic C2Hx. Understanding the complex, context-specific interactions between chronic SCI and CIH and how this ultimately impacts phrenic motor plasticity is important as we leverage AIH-induced motor plasticity to restore breathing and other non-respiratory motor functions in people with chronic SCI.
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Hipóxia , Neurônios Motores , Nervo Frênico , Receptores de Serotonina , Traumatismos da Medula Espinal , Animais , Masculino , Ratos , Medula Cervical/lesões , Medula Cervical/metabolismo , Vértebras Cervicais , Doença Crônica , Hipóxia/metabolismo , Neurônios Motores/metabolismo , Plasticidade Neuronal/fisiologia , Nervo Frênico/metabolismo , Ratos Sprague-Dawley , Receptores de Serotonina/metabolismo , Receptores de Serotonina/biossíntese , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Duchenne muscular dystrophy (DMD) is a devastating monogenic skeletal muscle-wasting disorder. Although many pharmacological and genetic interventions have been reported in preclinical studies, few have progressed to clinical trials with meaningful benefit. Identifying therapeutic potential can be limited by availability of suitable preclinical mouse models. More rigorous testing across models with varied background strains and mutations can identify treatments for clinical success. Here, we report the generation of a DMD mouse model with a CRISPR-induced deletion within exon 62 of the dystrophin gene (Dmd) and the first generated in BALB/c mice. Analysis of mice at 3, 6 and 12â months of age confirmed loss of expression of the dystrophin protein isoform Dp427 and resultant dystrophic pathology in limb muscles and the diaphragm, with evidence of centrally nucleated fibers, increased inflammatory markers and fibrosis, progressive decline in muscle function, and compromised trabecular bone development. The BALB/c.mdx62 mouse is a novel model of DMD with associated variations in the immune response and muscle phenotype, compared with those of existing models. It represents an important addition to the preclinical model toolbox for developing therapeutic strategies.