Neonatal intestinal colonization of Streptococcus agalactiae and the multiple modes of protection limiting translocation.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a predominant pathogen of neonatal sepsis, commonly associated with early-onset neonatal sepsis. GBS has also been associated with cases of late-onset sepsis potentially originating from the intestine. Previous findings have shown GBS can colonize the infant intestinal tract as part of the neonatal microbiota. To better understand GBS colonization dynamics in the neonatal intestine, we collected stool and milk samples from prematurely born neonates for identification of potential pathogens in the neonatal intestinal microbiota. GBS was present in approximately 10% of the cohort, and this colonization was not associated with maternal GBS status, delivery route, or gestational weight. Interestingly, we observed the relative abundance of GBS in the infant stool negatively correlated with maternal IgA concentration in matched maternal milk samples. Using a preclinical murine model of GBS infection, we report that both vertical transmission and direct oral introduction resulted in intestinal colonization of GBS; however, translocation beyond the intestine was limited. Finally, vaccination of dams prior to breeding induced strong immunoglobulin responses, including IgA responses, which were associated with reduced mortality and GBS intestinal colonization. Taken together, we show that maternal IgA may contribute to infant immunity by limiting the colonization of GBS in the intestine.

Streptococcus agalactiae and Escherichia coli induce distinct effector γδ T cell responses during neonatal sepsis.

Neonates born prematurely are vulnerable to life-threatening conditions such as bacterial sepsis. Streptococcus agalactiae (GBS) and Escherichia coli are frequent causative pathogens of neonatal sepsis, however, it remains unclear if these pathogens induce differential immune responses. We find that γδ T cells rapidly respond to single-organism GBS and E. coli bloodstream infections in neonatal mice. Furthermore, GBS and E. coli induce distinct cytokine production from IFN-γ and IL-17 producing γδ T cells, respectively. We also find that IL-17 production during E. coli infection is driven by γδTCR signaling, whereas IFN-γ production during GBS infection occurs independently of γδTCR signaling. The divergent effector responses of γδ T cells during GBS and E. coli infections impart distinctive neuroinflammatory phenotypes on the neonatal brain. Thus, the neonatal adaptive immune system differentially responds to distinct bacterial stimuli, resulting in unique neuroinflammatory phenotypes.

Streptococcus agalactiae and Escherichia coli Induce Distinct Effector γδ T Cell Responses During Neonatal Sepsis.

Neonates born prematurely are highly vulnerable to life-threatening conditions such as bacterial sepsis. Streptococcus agalactiae, also known as group B Streptococcus (GBS) and Escherichia coli are frequent causative pathogens of neonatal sepsis, however, it remains unclear if distinct sepsis pathogens induce differential adaptive immune responses. In the present study, we find that γδ T cells in neonatal mice rapidly respond to single-organism GBS and E. coli bloodstream infections and that these pathogens induce distinct activation and cytokine production from IFN-γ and IL-17 producing γδ T cells, respectively. We also report differential reliance on γδTCR signaling to elicit effector cytokine responses during neonatal sepsis, with IL-17 production during E. coli infection being driven by γδTCR signaling, and IFN-γ production during GBS infection occurring independently of γδTCR signaling. Furthermore, we report that the divergent effector responses of γδ T cells during GBS and E. coli infections impart distinctive neuroinflammatory phenotypes on the neonatal brain. The present study reveals that the neonatal adaptive immune system differentially responds to distinct bacterial stimuli, resulting in unique neuroinflammatory phenotypes.

Brain resident memory T cells rapidly expand and initiate neuroinflammatory responses following CNS viral infection.

The contribution of circulating verses tissue resident memory T cells (TRMs) to clinical neuropathology is an enduring question due to a lack of mechanistic insights. The prevailing view is TRMs are protective against pathogens in the brain. However, the extent to which antigen-specific TRMs induce neuropathology upon reactivation is understudied. Using the described phenotype of TRMs, we found that brains of naïve mice harbor populations of CD69+ CD103- T cells. Notably, numbers of CD69+ CD103- TRMs rapidly increase following neurological insults of various origins. This TRM expansion precedes infiltration of virus antigen-specific CD8 T cells and is due to proliferation of T cells within the brain. We next evaluated the capacity of antigen-specific TRMs in the brain to induce significant neuroinflammation post virus clearance, including infiltration of inflammatory myeloid cells, activation of T cells in the brain, microglial activation, and significant blood brain barrier disruption. These neuroinflammatory events were induced by TRMs, as depletion of peripheral T cells or blocking T cell trafficking using FTY720 did not change the neuroinflammatory course. Depletion of all CD8 T cells, however, completely abrogated the neuroinflammatory response. Reactivation of antigen-specific TRMs in the brain also induced profound lymphopenia within the blood compartment. We have therefore determined that antigen-specific TRMs can induce significant neuroinflammation, neuropathology, and peripheral immunosuppression. The use of cognate antigen to reactivate CD8 TRMs enables us to isolate the neuropathologic effects induced by this cell type independently of other branches of immunological memory, differentiating this work from studies employing whole pathogen re-challenge. This study also demonstrates the capacity for CD8 TRMs to contribute to pathology associated with neurodegenerative disorders and long-term complications associated with viral infections. Understanding functions of brain TRMs is crucial in investigating their role in neurodegenerative disorders including MS, CNS cancers, and long-term complications associated with viral infections including COVID-19.

Anti-PD-1 and Extended Half-life IL2 Synergize for Treatment of Murine Glioblastoma Independent of Host MHC Class I Expression.

Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti-PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I-restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I-independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.

Microglia and Perivascular Macrophages Act as Antigen Presenting Cells to Promote CD8 T Cell Infiltration of the Brain.

CD8 T cell infiltration of the central nervous system (CNS) is necessary for host protection but contributes to neuropathology. Antigen presenting cells (APCs) situated at CNS borders are thought to mediate T cell entry into the parenchyma during neuroinflammation. The identity of the CNS-resident APC that presents antigen via major histocompatibility complex (MHC) class I to CD8 T cells is unknown. Herein, we characterize MHC class I expression in the naïve and virally infected brain and identify microglia and macrophages (CNS-myeloid cells) as APCs that upregulate H-2Kb and H-2Db upon infection. Conditional ablation of H-2Kb and H-2Db from CNS-myeloid cells allowed us to determine that antigen presentation via H-2Db, but not H-2Kb, was required for CNS immune infiltration during Theiler's murine encephalomyelitis virus (TMEV) infection and drives brain atrophy as a consequence of infection. These results demonstrate that CNS-myeloid cells are key APCs mediating CD8 T cell brain infiltration.

Regulation of oral antigen delivery early in life: Implications for oral tolerance and food allergy.

The increasing incidence of food allergy remains a significant public health concern. Food allergy is partially due to a lack, or loss of tolerance to food allergens. Clinical outcomes surrounding early life practices, such as breastfeeding, antibiotic use and food allergen exposure, indicate the first year of life in children represents a unique time for shaping the immune system to reduce allergic outcomes. Animal models have identified distinctive aspects of when and where dietary antigens are delivered within the intestinal tract to promote oral tolerance prior to weaning. Additionally, animal models have identified contributions from maternal proteins from breast milk and bacterial products from the gut microbiota in regulating dietary antigen exposure and promoting oral tolerance, thus connecting decades of clinical observations on the benefits of breastfeeding, early food allergen introduction and antibiotic avoidance in the first year of life in reducing allergic outcomes. Here, we discuss how exposure to gut luminal antigens, including food allergens, is regulated in early life to generate protective tolerance and the implications of this process for preventing and treating food allergies.

Brain cancer induces systemic immunosuppression through release of non-steroid soluble mediators.

Immunosuppression of unknown aetiology is a hallmark feature of glioblastoma and is characterized by decreased CD4 T-cell counts and downregulation of major histocompatibility complex class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for glioblastoma. We recapitulated the immunosuppression observed in glioblastoma patients in the C57BL/6 mouse and investigated the aetiology of low CD4 T-cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of brain cancer, including mice harbouring GL261 glioma, B16 melanoma, and in a spontaneous model of diffuse intrinsic pontine glioma. In addition to thymic involution, we determined that tumour growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC II expression on blood leucocytes, and a modest increase in bone marrow resident CD4 T cells. Using parabiosis we report that thymic involution, declines in peripheral T-cell counts, and reduced major histocompatibility complex class II expression levels were mediated through circulating blood-derived factors. Conversely, T-cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is non-steroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the immunosuppression was not unique to cancer itself, but rather occurs in response to brain injury. Non-cancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that brain cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.

Conditional Silencing of H-2Db Class I Molecule Expression Modulates the Protective and Pathogenic Kinetics of Virus-Antigen-Specific CD8 T Cell Responses during Theiler's Virus Infection.

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS is cleared in C57BL/6 mice by a CD8 T cell response restricted by the MHC class I molecule H-2Db The identity and function of the APC(s) involved in the priming of this T cell response is (are) poorly defined. To address this gap in knowledge, we developed an H-2Db LoxP-transgenic mouse system using otherwise MHC class I-deficient C57BL/6 mice, thereby conditionally ablating MHC class I-restricted Ag presentation in targeted APC subpopulations. We observed that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2121-130 Loss of H-2Db on CD11c+ APCs mitigates the CD8 T cell response, preventing early viral clearance and immunopathology associated with CD8 T cell activity in the CNS. In contrast, animals with H-2Db-deficient LysM+ APCs retained early priming of Db:VP2121-130 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model enabling the critical dissection of H-2Db-restricted Ag presentation to CD8 T cells, revealing cell-specific and temporal features involved in the generation of CD8 T cell responses. Employing this novel system, we establish CD11c+ cells as pivotal to the establishment of acute antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2121-130, with functional implications both for T cell-mediated viral control and immunopathology.

Ketogenic diet treatment of children in the intensive care unit: Safety, tolerability, and effectiveness.

PURPOSE: The ketogenic diet (KD) is initiated emergently in the intensive care unit (ICU) for patients with super refractory status epilepticus (SRSE) and epileptic encephalopathies (EE). However, few data are available regarding safety, effectiveness, and long-term outcomes. METHODS: We performed a retrospective cohort study of consecutive patients with KD initiated in the ICU from 2010 to 2018 for SRSE and EE. We characterized time to ketosis, adverse effects, and seizure outcomes. Responders were defined as having ≥50 % reduction in seizure frequency compared to prior to KD initiation. RESULTS: We identified 29 patients. KD was initiated for SRSE in 12 patients, EE in 8 patients, and EE with SRSE in 9 patients. KD was initiated after a median of 9 days. Ketosis was achieved 2 days faster in fasted patients (p < 0.0001). All patients had at least 1 KD-related adverse effect, most often hypoglycemia, constipation, or acidosis. There was ≥50 % reduction in seizure frequency compared to prior to KD initiation by 1 week in 17/28 patients, seizure-freedom by 2 weeks in 7/28 patients, and weaned off anesthetics in 11/17 patients. All KD-responders at 1 month had continued response at 6 months. Mortality at 1 year was 24 %. There was no difference in KD response or mortality between KD indication groups. CONCLUSION: Emergent KD initiation in the ICU is feasible, safe, and often effective for SRSE and EE. Expected adverse effects were common but treatable. Morbidity and mortality in this group was high. A ≥ 50 % reduction in seizure is achieved in most responders by 1-2 weeks.

Comprehensive Migraine Initiative in the Pediatric Emergency Department Improves Treatment Outcomes.

OBJECTIVE: To compare pediatric migraine treatment efficacy in the emergency department before and after the implementation of a comprehensive migraine initiative, consisting of a standardized treatment protocol, provider educational series and standardized physician documentation template. BACKGROUND: Pediatric migraine is common, accounting for 1% of pediatric emergency department visits. Yet there is large variability in treatment practices, with few studies looking into measures of both clinical effectiveness and timeliness of treatment following implementation of standardized protocols. METHODS: A single-center retrospective chart review of pediatric patients presenting to the emergency department with migraine before and after implementation of an institutional headache initiative designed to more effectively and efficiently deliver care to pediatric migraine patients. RESULTS: The study yielded 110 patients each in the intervention and preintervention groups. There were no significant differences in patient characteristics with respect to age, gender, or initial pain score. Compared with the preintervention group, the intervention group demonstrated a significant reduction in headache pain score prior to discharge (decrease of 5.9 vs 4.8 in preintervention group, P value .006) with a greater percentage of patients achieving ≥50% reduction in pain (82% vs 67% in preintervention group, P value .039). Additionally, we found a significantly decreased time to treatment in the intervention group compared with the preintervention group (1.8 vs 2.1 hours, P value .046). CONCLUSION: Through the use of a standardized treatment protocol, improved provider education, and ease of documentation, this comprehensive migraine initiative improved efficacy and efficiency of migraine treatment in the pediatric emergency department.

The probability of seizures during continuous EEG monitoring in high-risk neonates.

OBJECTIVE: We evaluated the impact of monitoring indication, early electroencephalography (EEG), and clinical features on seizure risk in all neonates undergoing continuous EEG (cEEG) monitoring following a standardized monitoring protocol. METHODS: All cEEGs from unique neonates 34-48 weeks postmenstrual age monitored from 1/2011-10/2017 (n = 291) were included. We evaluated the impact of cEEG monitoring indication (acute neonatal encephalopathy [ANE], suspicious clinical events [SCEs], or other high-risk conditions [OHRs]), age, medication status, and early EEG abnormalities (including the presence of epileptiform discharges and abnormal background continuity, amplitude, asymmetry, asynchrony, excessive sharp transients, and burst suppression) on time to first seizure and overall seizure risk using Kaplan-Meier survival curves and multivariable Cox proportional hazards models. RESULTS: Seizures occurred in 28% of high-risk neonates. Discontinuation of monitoring after 24 hours of seizure-freedom would have missed 8.5% of neonates with seizures. Overall seizure risk was lower in neonates monitored for ANE compared to OHR (P = .004) and trended lower compared to SCE (P = .097). The time course of seizure presentation varied by group, where the probability of future seizure was less than 1% after 17 hours of seizure-free monitoring in the SCE group, but required 42 hours in the OHR group, and 73 hours in the ANE group. The presence of early epileptiform discharges increased seizure risk in each group (ANE: adjusted hazard ratio [aHR] 4.32, 95% confidence interval [CI] 1.23-15.13, P = .022; SCE: aHR 10.95, 95% CI 4.77-25.14, P < 1e-07; OHR: aHR 56.90, 95% CI 10.32-313.72, P < 1e-05). SIGNIFICANCE: Neonates who undergo cEEG are at high risk for seizures, and risk varies by monitoring indication and early EEG findings. Seizures are captured in nearly all neonates undergoing monitoring for SCE within 24 hours of cEEG monitoring. Neonates monitored for OHR and ANE can present with delayed seizures and require longer durations of monitoring. Early epileptiform discharges are the best early EEG feature to predict seizure risk.

Functional consequences of 17q21.31/WNT3-WNT9B amplification in hPSCs with respect to neural differentiation.

Human pluripotent stem cell (hPSC) lines exhibit repeated patterns of genetic variation, which can alter in vitro properties as well as suitability for clinical use. We examined associations between copy-number variations (CNVs) on chromosome 17 and hPSC mesodiencephalic dopaminergic (mDA) differentiation. Among 24 hPSC lines, two karyotypically normal lines, BG03 and CT3, and BG01V2, with trisomy 17, exhibited amplification of the WNT3/WNT9B region and rapid mDA differentiation. In hPSC lines with amplified WNT3/WNT9B, basic fibroblast growth factor (bFGF) signaling through mitogen-activated protein kinase (MAPK)/ERK amplifies canonical WNT signaling by phosphorylating LRP6, resulting in enhanced undifferentiated proliferation. When bFGF is absent, noncanonical WNT signaling becomes dominant due to upregulation of SIAH2, enhancing JNK signaling and promoting loss of pluripotency. When bFGF is present during mDA differentiation, stabilization of canonical WNT signaling causes upregulation of LMX1A and mDA induction. Therefore, CNVs in 17q21.31, a "hot spot" for genetic variation, have multiple and complex effects on hPSC cellular phenotype.

Is there an ideal way to discontinue the ketogenic diet?

It is unclear what the ideal weaning speed of the ketogenic diet should be and the resultant risk of seizure worsening. A retrospective chart review was performed of children who discontinued the ketogenic diet at Johns Hopkins Hospital from January 2000 to June 2010. Speed of discontinuation was categorized into immediate (<1 week), quick (1-6 weeks), or slow (>6 weeks) rates. One hundred and eighty-three children were identified. Children with both a longer diet duration (p=0.004) and lower seizure frequency (p<0.001) were weaned more slowly by our group. There was no significant difference in the incidence of seizures worsening between discontinuation rates. However, there was an increased risk of seizures worsening in those specifically with a 50-99% seizure reduction (30% vs. 8%, p<0.0001) and for that level of seizure improvement, in those who were receiving more anticonvulsants (1.4 vs. 0.8, p=0.01). In summary, there does not appear to be an increased risk of seizure exacerbation with rapid ketogenic diet discontinuations. Those who improved 50-99% and were receiving more anticonvulsants were at the highest risk overall. Discontinuing the ketogenic diet over weeks rather than months appears safe.

Cocaine causes deficits in radial migration and alters the distribution of glutamate and GABA neurons in the developing rat cerebral cortex.

Prenatal cocaine exposure induces cytoarchitectural changes in the embryonic neocortex; however, the biological mechanisms and type of cortical neurons involved in these changes are not known. Previously, we found that neural progenitor proliferation in the neocortical ventricular zone (VZ) is inhibited by cocaine; here, we examine the changes in cortical neurogenesis and migration of glutamate and GABA neurons induced by prenatal cocaine exposure. Pregnant rats received 20 mg/kg of cocaine intraperitoneally twice at an interval of 12 h during three periods of neocortical neurogenesis. Neocortical area and distribution of developing neurons were examined by counting Tuj1+, glutamate+, or GABA+ cells in different areas of the cerebral cortex. Cocaine decreased neocortical area by reducing the size of the Tuj1+ layer, but only when administered during early periods of neocortical neurogenesis. The number of glutamatergic neurons was increased in the VZ but was decreased in the outer cortical laminae. Although the number of GABA+ neurons in the VZ of both the neocortex and ganglionic eminences was unchanged, GABA+ cells decreased in all other neocortical laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. These findings suggest that in utero cocaine exposure disturbs radial migration of neocortical neurons, possibly because of decreased radial glia guiding support through enhanced differentiation of neocortical VZ progenitors. Cocaine interrupts radial migration of both glutamatergic and GABAergic neurons within the neocortex, in addition to the tangential migration of GABAergic neurons from the subcortical telecephalon. This may result in abnormal neocortical cytoarchitecture and concomitant adverse functional effects.

The adenosine A2A antagonist MSX-3 reverses the effort-related effects of dopamine blockade: differential interaction with D1 and D2 family antagonists.

RATIONALE: Brain dopamine (DA) participates in the modulation of instrumental behavior, including aspects of behavioral activation and effort-related choice behavior. Rats with impaired DA transmission reallocate their behavior away from food-seeking behaviors that have high response requirements, and instead select less effortful alternatives. Although accumbens DA is considered a critical component of the brain circuitry regulating effort-related choice behavior, emerging evidence demonstrates a role for adenosine A(2A) receptors. OBJECTIVE: Adenosine A(2A) receptor antagonism has been shown to reverse the effects of DA antagonism. The present experiments were conducted to determine if this effect was dependent upon the subtype of DA receptor that was antagonized to produce the changes in effort-related choice. MATERIALS AND METHODS: The adenosine A(2A) receptor antagonist MSX-3 (0.5-2.0 mg/kg IP) was assessed for its ability to reverse the effects of the D1 family antagonist SCH39166 (ecopipam; 0.2 mg/kg IP) and the D2 family antagonist eticlopride (0.08 mg/kg IP), using a concurrent lever pressing/chow feeding procedure. RESULTS: MSX-3 produced a substantial dose-related reversal of the effects of eticlopride on lever pressing and chow intake. At the highest dose of MSX-3, there was a complete reversal of the effects of eticlopride on lever pressing. In contrast, MSX-3 produced only a minimal attenuation of the effects of SCH39166, as measured by regression and effect size analyses. CONCLUSIONS: The greater ability of MSX-3 to reverse the effects of D2 vs. D1 blockade may be related to the colocalization of D2 and adenosine A(2A) receptors on the same population of striatal neurons.

Nucleus accumbens adenosine A2A receptors regulate exertion of effort by acting on the ventral striatopallidal pathway.

Goal-directed actions are sensitive to work-related response costs, and dopamine in nucleus accumbens is thought to modulate the exertion of effort in motivated behavior. Dopamine-rich striatal areas such as nucleus accumbens also contain high numbers of adenosine A(2A) receptors, and, for that reason, the behavioral and neurochemical effects of the adenosine A(2A) receptor agonist CGS 21680 [2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine] were investigated. Stimulation of accumbens adenosine A(2A) receptors disrupted performance of an instrumental task with high work demands (i.e., an interval lever-pressing schedule with a ratio requirement attached) but had little effect on a task with a lower work requirement. Immunohistochemical studies revealed that accumbens neurons that project to the ventral pallidum showed adenosine A(2A) receptors immunoreactivity. Moreover, activation of accumbens A(2A) receptors by local injections of CGS 21680 increased extracellular GABA levels in the ventral pallidum. Combined contralateral injections of CGS 21680 into the accumbens and the GABA(A) agonist muscimol into ventral pallidum (i.e., "disconnection" methods) also impaired response output, indicating that these structures are part of a common neural circuitry regulating the exertion of effort. Thus, accumbens adenosine A(2A) receptors appear to regulate behavioral activation and effort-related processes by modulating the activity of the ventral striatopallidal pathway. Research on the effort-related functions of these forebrain systems may lead to a greater understanding of pathological features of motivation, such as psychomotor slowing, anergia, and fatigue in depression.

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Sla1p is a functionally modular component of the yeast cortical actin cytoskeleton required for correct localization of both Rho1p-GTPase and Sla2p, a protein with talin homology.

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme beta-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls. Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.