High-fat diet, microbiome-gut-brain axis signaling, and anxiety-like behavior in male rats.

Obesity, associated with the intake of a high-fat diet (HFD), and anxiety are common among those living in modern urban societies. Recent studies suggest a role of microbiome-gut-brain axis signaling, including a role for brain serotonergic systems in the relationship between HFD and anxiety. Evidence suggests the gut microbiome and the serotonergic brain system together may play an important role in this response. Here we conducted a nine-week HFD protocol in male rats, followed by an analysis of the gut microbiome diversity and community composition, brainstem serotonergic gene expression (tph2, htr1a, and slc6a4), and anxiety-related defensive behavioral responses. We show that HFD intake decreased alpha diversity and altered the community composition of the gut microbiome in association with obesity, increased brainstem tph2, htr1a and slc6a4 mRNA expression, including in the caudal part of the dorsomedial dorsal raphe nucleus (cDRD), a subregion previously associated with stress- and anxiety-related behavioral responses, and, finally, increased anxiety-related defensive behavioral responses. The HFD increased the Firmicutes/Bacteroidetes ratio relative to control diet, as well as higher relative abundances of Blautia, and decreases in Prevotella. We found that tph2, htr1a and slc6a4 mRNA expression were increased in subregions of the dorsal raphe nucleus in the HFD, relative to control diet. Specific bacterial taxa were associated with increased serotonergic gene expression in the cDRD. Thus, we propose that HFD-induced obesity is associated with altered microbiome-gut-serotonergic brain axis signaling, leading to increased anxiety-related defensive behavioral responses in rats.

Postoperative Interventions in Pediatric Digital Replantation: A Tertiary Referral Center Case Review.

PURPOSE: Although a few case series have been published describing the excellent outcomes of replantation and revascularization operations in children, there has been limited study of the hospital course that these patients experience and the number of potentially harmful interventions and treatments that occur. The purpose of this study was to detail the results of various postoperative interventions, including anticoagulation, transfusion, leeching, sedation, and additional anesthetic exposures. METHODS: Twenty-nine patients aged less than 18 years had 34 digital revascularizations or replantations performed between January 2000 and May 2020. The details of each patient's presentation, surgery, and postoperative care were analyzed. RESULTS: Nine of 29 children underwent repeat anesthetics, including 6 revision amputations. No demographic, surgical, or postoperative variables consistently preceded revision amputation or additional anesthetic procedures. Only 5 patients had >1 hemoglobin (Hb) measurement. Two patients received blood transfusions; the average drop in Hb was 3.5 g/dL from before surgery to the lowest after surgery. Four patients underwent leech therapy. Only patients receiving leech therapy required postoperative transfusions. Anticoagulation regimens were prescribed on the basis of demographic and surgical factors, although no medication or regimen seemed to affect outcomes. CONCLUSIONS: Although the experience of digital replantation is essentially the same in pediatric patients as adults, there may be different ramifications for children. Specifically, postoperative management of pediatric digital replantation or revascularization can involve multiple interventions that carry their risks. Parents should be counseled about the risks of anticoagulants, transfusions, and repeat anesthetics, and clinicians should monitor Hb closely when using leech therapy. TYPE OF STUDY/LEVEL OF EVIDENCE: Case series, Therapeutic IV.

Fungal Pyelonephritis and Fungemia Due to Obstructive Uropathy.

INTRODUCTION: Funguria is often a benign and common occurrence in the hospital. However, invasive fungal pyelonephritis due to obstructive uropathy is uncommon and can be difficult to treat. Typically, there are 2 mechanisms by which Candida albicans infects the upper urinary tract: by ascending from the lower urinary tract or via hematogenous spread to the kidneys. CASE PRESENTATION: We present a case of fungal pyelonephritis, likely due to obstructive uropathy, leading to fungemia in a 70-year-old man who had a recent history of colovesicular fistula and indwelling foley catheter. DISCUSSION: The patient had many identified risk factors contributing to the development of fungal pyelonephritis, including diabetes mellitus and structural urinary tract aberrancies, which were further complicated by his recent colovesicular fistula and repair. CONCLUSION: Although fungal pyelonephritis with fungemia is relatively rare, it should not be excluded from differential diagnostics. Despite a unique host of risk factors, a direct approach led to successful treatment.

Challenges and Potential in Targeted Muscle Reinnervation in Pediatric Amputees.

Targeted muscle reinnervation (TMR) is a powerful new tool in preventing and treating residual limb and phantom limb pain. In the adult population, TMR is rapidly becoming standard of care; however, there is a paucity of literature regarding indications and outcomes of TMR in the pediatric population. We present 2 cases of pediatric patients who sustained amputations and the relevant challenges associated with TMR in their cases. One is a 7-year-old patient who developed severe phantom and residual limb pain after a posttraumatic above-knee amputation. He failed pharmacologic measures and underwent TMR. He obtained complete relief of his symptoms and is continuing to do well 1.5 years postoperatively. The other is a 2-year-old boy with bilateral wrist and below-knee amputations as sequelae of sepsis. TMR was not performed because the patient never demonstrated evidence of phantom limb pain or symptomatic neuroma formation. We use these 2 cases to explore the challenges particular to pediatric patients when considering treatment with TMR, including capacity to report pain, risks of anesthesia, and cortical plasticity. These issues will be critical in determining how TMR will be applied to pediatric patients.

Case report: Spontaneous pneumothorax in resolved, uncomplicated COVID-19 Pneumonia-A literature review.

As the global COVID-19 pandemic has progressed, awareness of uncommon presentations and complications has increased. The actual incidence of spontaneous pneumothorax was found to be 0.66%, or six patients out of 902 who tested positive in recently published literature of 3368 patients (Zantah M, Dominguez Castillo E, Townsend R, Dikengil F, Criner GJ. Pneumothorax in COVID-19 disease-incidence and clinical characteristics. Respir Res. 2020 Sep 16; 21 (1):236.). Of those six patients, only two (0.22%) were not associated with mechanical ventilation barotrauma or comorbid lung disease such as COPD. Here, we present a spontaneous pneumothorax and pneumomediastinum in a patient four days after he had been discharged from hospitalization due to uncomplicated COVID-19 pneumonia.

Effects of Immunization With the Soil-Derived Bacterium Mycobacterium vaccae on Stress Coping Behaviors and Cognitive Performance in a "Two Hit" Stressor Model.

Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a "two hit" stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day -28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days -21, -14, -7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0-56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days -1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.

Effects of maternal separation on serotonergic systems in the dorsal and median raphe nuclei of adult male Tph2-deficient mice.

Previous studies have highlighted interactions between serotonergic systems and adverse early life experience as important gene x environment determinants of risk of stress-related psychiatric disorders. Evidence suggests that mice deficient in Tph2, the rate-limiting enzyme for brain serotonin synthesis, display disruptions in behavioral phenotypes relevant to stress-related psychiatric disorders. The aim of this study was to determine how maternal separation in wild-type, heterozygous, and Tph2 knockout mice affects mRNA expression of serotonin-related genes. Serotonergic genes studied included Tph2, the high-affinity, low-capacity, sodium-dependent serotonin transporter (Slc6a4), the serotonin type 1a receptor (Htr1a), and the corticosterone-sensitive, low-affinity, high-capacity sodium-independent serotonin transporter, organic cation transporter 3 (Slc22a3). Furthermore, we studied corticotropin-releasing hormone receptors 1 (Crhr1) and 2 (Crhr2), which play important roles in controlling serotonergic neuronal activity. For this study, offspring of Tph2 heterozygous dams were exposed to daily maternal separation for the first two weeks of life. Adult, male wild-type, heterozygous, and homozygous offspring were subsequently used for molecular analysis. Maternal separation differentially altered serotonergic gene expression in a genotype- and topographically-specific manner. For example, maternal separation increased Slc6a4 mRNA expression in the dorsal part of the dorsal raphe nucleus in Tph2 heterozygous mice, but not in wild-type or knockout mice. Overall, these data are consistent with the hypothesis that gene x environment interactions, including serotonergic genes and adverse early life experience, play an important role in vulnerability to stress-related psychiatric disorders.

Treatment with a heat-killed preparation of Mycobacterium vaccae after fear conditioning enhances fear extinction in the fear-potentiated startle paradigm.

The hygiene hypothesis or "Old Friends" hypothesis proposes that inflammatory diseases are increasing in modern urban societies, due in part to reduced exposure to microorganisms that drive immunoregulatory circuits and a failure to terminate inappropriate inflammatory responses. Inappropriate inflammation is also emerging as a risk factor for anxiety disorders, affective disorders, and trauma-and stressor-related disorders, including posttraumatic stress disorder (PTSD), which is characterized as persistent re-experiencing of the trauma after a traumatic experience. Traumatic experiences can lead to long-lasting fear memories and fear potentiation of the acoustic startle reflex. The acoustic startle reflex is an ethologically relevant reflex and can be potentiated in both humans and rats through Pavlovian conditioning. Mycobacterium vaccae is a soil-derived bacterium with immunoregulatory and anti-inflammatory properties that has been demonstrated to enhance fear extinction in the fear-potentiated startle paradigm when given prior to fear conditioning. To determine if immunization with M. vaccae after fear conditioning also has protective effects, adult male Sprague Dawley rats underwent fear conditioning on days -37 and -36 followed by immunizations (3x), once per week beginning 24 h following fear conditioning, with a heat-killed preparation of M. vaccae NCTC 11659 (0.1 mg, s.c., in 100 µl borate-buffered saline) or vehicle, and, then, 3 weeks following the final immunization, were tested in the fear-potentiated startle paradigm (n = 12 per group). Rats underwent fear extinction training on days 1 through 6 followed by spontaneous recovery 14 days later (day 20). Rats were euthanized on day 21 and brain tissue was sectioned for analysis of Tph2, Htr1a, Slc6a4, Slc22a3, and Crhr2 mRNA expression throughout the brainstem dorsal and median raphe nuclei. Immunization with M. vaccae did not affect fear expression on day 1. However, M. vaccae-immunized rats showed enhanced enhanced within-session fear extinction on day 1 and enhanced between-session fear extinction beginning on day 2, relative to vehicle-immunized controls. Immunization with M. vaccae and fear-potentiated startle had minimal effects on serotonergic gene expression when assessed 42 days after the final immunization. Together with previous studies, these data are consistent with the hypothesis that immunoregulatory strategies, such as immunization with M. vaccae, have potential for both prevention and treatment of trauma- and stressor-related psychiatric disorders.

Evidence that preimmunization with a heat-killed preparation of Mycobacterium vaccae reduces corticotropin-releasing hormone mRNA expression in the extended amygdala in a fear-potentiated startle paradigm.

Posttraumatic stress disorder (PTSD) is a trauma and stressor-related disorder that is characterized by dysregulation of glucocorticoid signaling, chronic low-grade inflammation, and impairment in the ability to extinguish learned fear. Corticotropin-releasing hormone (Crh) is a stress- and immune-responsive neuropeptide secreted from the paraventricular nucleus of the hypothalamus (PVN) to stimulate the hypothalamic-pituitary-adrenal (HPA) axis; however, extra-hypothalamic sources of Crh from the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST) govern specific fear- and anxiety-related defensive behavioral responses. We previously reported that preimmunization with a heat-killed preparation of the immunoregulatory environmental bacterium Mycobacterium vaccae NCTC 11659 enhances fear extinction in a fear-potentiated startle (FPS) paradigm. In this follow-up study, we utilized an in situ hybridization histochemistry technique to investigate Crh, Crhr1, and Crhr2 mRNA expression in the CeA, BNST, and PVN of the same rats from the original study [Fox et al., 2017, Brain, Behavior, and Immunity, 66: 70-84]. Here, we demonstrate that preimmunization with M. vaccae NCTC 11659 decreases Crh mRNA expression in the CeA and BNST of rats exposed to the FPS paradigm, and, further, that Crh mRNA expression in these regions is correlated with fear behavior during extinction training. These data are consistent with the hypothesis that M. vaccae promotes stress-resilience by attenuating Crh production in fear- and anxiety-related circuits. These data suggest that immunization with M. vaccae may be an effective strategy for prevention of fear- and anxiety-related disorders.

Two models of inescapable stress increase tph2 mRNA expression in the anxiety-related dorsomedial part of the dorsal raphe nucleus.

Expression of TPH2, the rate-limiting enzyme for brain serotonin synthesis, is elevated in the dorsal raphe nucleus (DR) of depressed suicide victims. One hypothesis is that this increase in TPH2 expression is stress-induced. Here, we used an established animal model to address whether exposure to an acute stressor, inescapable tail shock (IS), increases tph2 mRNA and Tph2 protein expression, and if IS sensitizes the DR to a subsequent, heterotypic stressor. In Experiment 1, we measured tph2 mRNA expression 4 h after IS or home cage (HC) control conditions in male rats, using in situ hybridization histochemistry. In Experiment 2, we measured Tph2 protein expression 12 h or 24 h after IS using western blot. In Experiment 3, we measured tph2 mRNA expression following IS on Day 1, and cold swim stress (10 min, 15 °C) on Day 2. Inescapable tail shock was sufficient to increase tph2 mRNA expression 4 h and 28 h later, selectively in the dorsomedial DR (caudal aspect of the dorsal DR, cDRD; an area just rostral to the caudal DR, DRC) and increased Tph2 protein expression in the DRD (rostral and caudal aspects of the dorsal DR combined) 24 h later. Cold swim increased tph2 mRNA expression in the dorsomedial DR (cDRD) 4 h later. These effects were associated with increased immobility during cold swim, elevated plasma corticosterone, and a proinflammatory plasma cytokine milieu (increased interleukin (IL)-6, decreased IL-10). Our data demonstrate that two models of inescapable stress, IS and cold swim, increase tph2 mRNA expression selectively in the anxiety-related dorsomedial DR (cDRD).

Cooperation and competition in mismatch repair: very short-patch repair and methyl-directed mismatch repair in Escherichia coli.

In Escherichia coli and related enteric bacteria, repair of base-base mismatches is performed by two overlapping biochemical processes, methyl-directed mismatch repair (MMR) and very short-patch (VSP) repair. While MMR repairs replication errors, VSP repair corrects to C*G mispairs created by 5-methylcytosine deamination to T. The efficiency of the two pathways changes during the bacterial life cycle; MMR is more efficient during exponential growth and VSP repair is more efficient during the stationary phase. VSP repair and MMR share two proteins, MutS and MutL, and although the two repair pathways are not equally dependent on these proteins, their dual use creates a competition within the cells between the repair processes. The structural and biochemical data on the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays a role similar to MutH (also an endonuclease) in MMR. Biochemical and genetic studies of the two repair pathways have helped eliminate certain models for MMR and put restrictions on models that can be developed regarding either repair process. We review here recent information about the biochemistry of both repair processes and describe the balancing act performed by cells to optimize the competing processes during different phases of the bacterial life cycle.