Rabbits were eyeblink conditioned while their accessory abducens nucleus (ACC), facial nucleus (FN), and surrounding reticular formation (RF) were temporarily inactivated with microinjections of muscimol to determine whether these structures are critically involved in acquisition of the conditioned eyeblink response (CR). Rabbits performed no CRs or unconditioned responses (URs) during inactivation training. Training was continued without inactivation and rabbits performed the CR at asymptotic levels from the start of training without inactivation. They had fully learned the CR while their ACC, FN, and RF were inactivated, despite performing no CRs or URs at all during inactivation. These results rule out any critical role for neurons within the ACC, FN, or surrounding RF in acquisition of the classically conditioned eyeblink response.
spp. are Gram-positive opportunistic pathogens that affect largely immunocompromised patients, leading to serious pulmonary or systemic infections. Combination therapy using the folate biosynthesis pathway inhibitors trimethoprim (TMP) and sulfamethoxazole (SMX) is commonly used as an antimicrobial therapy. Not surprisingly, as antibiotic therapies for nocardiosis can extend for many months, resistance to TMP-SMX has emerged. Using experimental evolution, we surveyed the genetic basis of adaptation to TMP-SMX across 8 strains of and 2 strains of By employing both continuous experimental evolution to provide longitudinal information on the order of changes and characterization of resistant endpoint isolates, we observe changes that are consistent with modifications of two enzymes of the folate biosynthesis pathway: dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) (FolP), with the mutations often being clustered near the active site of the enzymes. While changes to DHFR and DHPS might be expected, we also noted that mutations in a previously undescribed homolog of DHPS (DHPS2 or FolP2) that was annotated as being "nonfunctional" were also sufficient to generate TMP-SMX resistance, which serves as a cautionary tale for the use of automated annotation by investigators and for the future discovery of drugs against this genus. Additionally, overlapped glucosyl-3-phosphoglycerate synthase. Remarkably, an adaptive frameshift mutation within the overlapping region resulted in a new in-frame fusion to the downstream gene to produce a potentially new bifunctional enzyme. How a single potentially bifunctional DHPS2 enzyme might confer resistance is unclear. However, it highlights the unexpected ways in which adaptive evolution finds novel solutions for selection.
Recent advances in high‐throughput (HTP) automated mini‐bioreactor systems have significantly improved development timelines for early‐stage biologic programs. Automated platforms such as the ambr® 250 have demonstrated the ability, using appropriate scale‐down approaches, to provide reliable estimates of process performance and product quality from bench to pilot scale, but data sets comparing to large‐scale commercial processes (>10,000 L) are limited. As development moves toward late stages, specifically process characterization (PC), a qualified scale‐down model (SDM) of the commercial process is a regulatory requirement as part of Biologics License Application (BLA)‐enabling activities. This work demonstrates the qualification of the ambr® 250 as a representative SDM for two monoclonal antibody (mAb) commercial processes at scales >10,000 L. Representative process performance and product quality associated with each mAb were achieved using appropriate scale‐down approaches, and special attention was paid to pCO2 to ensure consistent performance and product quality. Principal component analysis (PCA) and univariate equivalence testing were utilized in the qualification of the SDM, along with a statistical evaluation of process performance and product‐quality attributes for comparability. The ambr® 250 can predict these two commercial‐scale processes (at center‐point condition) for cell‐culture performance and product quality. The time savings and resource advantages to performing PC studies in a small‐scale HTP system improves the potential for the biopharmaceutical industry to get products to patients more quickly.
Salmonella spp. express Salmonella pathogenicity island 1 Type III Secretion System 1 (T3SS-1) genes to mediate the initial phase of interaction with their host. Prior studies indicate short-chain fatty acids, microbial metabolites at high concentrations in the gastrointestinal tract, limit population-level T3SS-1 gene expression. However, only a subset of Salmonella cells in a population express these genes, suggesting short-chain fatty acids could decrease T3SS-1 population-level expression by acting on per-cell expression or the proportion of expressing cells. Here, we combine single-cell, theoretical, and molecular approaches to address the effect of short-chain fatty acids on T3SS-1 expression. Our in vitro results show short-chain fatty acids do not repress T3SS-1 expression by individual cells. Rather, these compounds act to selectively slow the growth of T3SS-1–expressing cells, ultimately decreasing their frequency in the population. Further experiments indicate slowed growth arises from short-chain fatty acid–mediated depletion of the proton motive force. By influencing the T3SS-1 cell-type proportions, our findings imply gut microbial metabolites act on cooperation between the two cell types and ultimately influence Salmonella’s capacity to establish within a host.
Patients with serious illnesses may experience existential and psychosocial distress contributing to their pain and suffering. Addressing existential distress is challenging and may require a multidisciplinary approach. Often, providers feel uncomfortable or ill equipped to care for patients suffering from this distress. In the sample case, the patient has a life-limiting disease and is concerned about his family forgetting him, experiencing loss of dignity and narrative foreclosure. Loss of dignity is sensing hopelessness and worthlessness and a loss of self-determination. Narrative foreclosure is the premature conviction that one's life story has effectively ended. Beneficial interventions include meaning-centered psychotherapy and dignity therapy (DT). Both have an underlying theme of attempting to reverse the narrative foreclosure for patients with serious illnesses and maintain a sense of meaning in life. In addition, patients can be referred to palliative care to enhance coping and decrease depressive symptoms. Dr. Harvey Chochinov has outlined a framework that clinicians can use to care for their patients in a compassionate manner to specifically combat meaninglessness. In DT, a generativity document is created for the patient and their loved ones as part of the treatment along with the opportunity to answer the dignity conserving question. Success of this route of intervention includes greater will to live, reductions in stress, and benefits perceived by family. This article aims to give a framework to treat patients with serious illnesses experiencing psychosocial and/or existential distress.
The dynamic immunological changes occurring throughout pregnancy are well-orchestrated and important for the success of the pregnancy. One of the key immune adaptations is the maternal immune tolerance towards the semi-allogeneic fetus. In this review, we provide a comprehensive overview of what is known about the innate and adaptive immunological changes in pregnancy and the role(s) of specific immune cells during physiological and pathological pregnancy. Alongside this, we provided details of remaining questions and challenges, as well as future perspectives for this growing field of research. Understanding the immunological changes that occur can inform potential strategies on treatments for the optimal health of the neonate and pregnant individual both during and after pregnancy.
Salmonella spp. express Salmonella pathogenicity island 1 (SPI-1) genes to mediate the initial phase of interaction with host cells. Prior studies indicate short-chain fatty acids, microbial metabolites at high concentrations in the gastrointestinal tract, limit SPI-1 gene expression. A number of reports show only a subset of Salmonella cells in a population express these genes, suggesting short-chain fatty acids could decrease SPI-1 population-level expression by acting on per-cell expression and/or the proportion of expressing cells. Here, we combine single-cell, theoretical, and molecular approaches to address the effect of short-chain fatty acids on SPI-1 expression. Our results show short-chain fatty acids do not repress SPI-1 expression by individual cells. Rather, these compounds act to selectively slow the growth of SPI-1 expressing cells, ultimately decreasing their frequency in the population. Further experiments indicate slowed growth arises from short-chain fatty acid-mediated depletion of the proton motive force. By influencing the SPI-1 cell-type proportions, our findings imply gut microbial metabolites act on cooperation between the two cell-types and ultimately influence the capacity of Salmonella to establish within a host.
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