TOR complex 1 negatively regulates NDR kinase Cbk1 to control cell separation in budding yeast.

The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying molecular mechanisms by which TOR might regulate events after anaphase remain unknown. We show for the first time that one of the 2 TOR complexes in budding yeast, TORC1, blocks the separation of cells following cytokinesis by phosphorylation of a member of the NDR (nuclear Dbf2-related) protein-kinase family, the protein Cbk1. We observe that TORC1 alters the phosphorylation pattern of Cbk1 and we identify a residue within Cbk1 activation loop, T574, for which a phosphomimetic substitution makes Cbk1 catalytically inactive and, indeed, reproduces TORC1 control over cell separation. In addition, we identify the exocyst component Sec3 as a key substrate of Cbk1, since Sec3 activates the SNARE complex to promote membrane fusion. TORC1 activity ultimately compromises the interaction between Sec3 and a t-SNARE component. Our data indicate that TORC1 negatively regulates cell separation in budding yeast by participating in Cbk1 phosphorylation, which in turn controls the fusion of secretory vesicles transporting hydrolase at the site of division.

Facile detection of peptide-protein interactions using an electrophoretic crosslinking shift assay.

Protein-protein interactions with high specificity and low affinity are functionally important but are not comprehensively understood because they are difficult to identify. Particularly intriguing are the dynamic and specific interactions between folded protein domains and short unstructured peptides known as short linear motifs (SLiMs). Such domain-motif interactions (DMIs) are often difficult to identify and study because affinities are modest to weak. Here we describe "electrophoretic crosslinking shift assay" (ECSA), a simple in vitro approach that detects transient, low affinity interactions by covalently crosslinking a prey protein and a fluorescently labeled bait. We demonstrate this technique on the well characterized DMI between MAP kinases and unstructured D-motif peptide ligands. We show that ECSA detects sequence-specific micromolar interactions using less than a microgram of input prey protein per reaction, making it ideal for verifying candidate low-affinity DMIs of components that purify with low yield. We propose ECSA as an intermediate step in SLiM characterization that bridges the gap between high throughput techniques such as phage display and more resource-intensive biophysical and structural analysis.

Does Completeness of the Mesorectal Excision Still Correlate With Local Recurrence?

BACKGROUND: Total mesorectal excision is the standard surgical procedure for rectal cancer treatment. Several studies have shown a close correlation between the prognosis of patients with rectal cancer and the completeness of the mesorectal specimen. OBJECTIVE: To assess the correlation between macroscopic assessment of mesorectal excision and long-term oncological outcomes. DESIGN: Retrospective analysis of an Institutional Review Board-approved database. SETTINGS: Tertiary referral center. PATIENTS: Patients with rectal cancer who were operated on between March 2016 and October 2019 were classified into 3 groups based on the mesorectal specimen quality: complete, near complete, and incomplete. Only patients with a follow-up of ≥2 years and without signs of preoperative distant disease were included. MAIN OUTCOME MEASURES: Relationship between total mesorectal excision and local and distant recurrence rates in patients with rectal cancer. RESULTS: A total of 124 patients (35.5% females) were included in the analysis, with a mean age of 58.1 (SD 12) years and a mean BMI of 26.4 (SD 4.59) kg/m². Neoadjuvant chemoradiation was administered to 71% of patients, whereas 13.7% received total neoadjuvant therapy. Restorative procedures were performed in 107 patients (86.3%), whereas 17 patients (13.7%) underwent abdominoperineal resection. The majority of mesorectal excision specimens (87.09%) were complete or near complete. Local recurrence rates were 6.3% (1/16) in the incomplete and 7.4% (8/108) in the complete/near complete group ( p = 0.86). Metachronous distant metastases occurred in 6 patients (37.5%) in the incomplete group and in 24 patients (22.2%) in the complete/near complete group (p = 0.18). Thus, specimen quality did not appear to impact disease-free survival. LIMITATIONS: Retrospective, single-center study with relatively short follow-up. CONCLUSIONS: In the era of a multidisciplinary approach and extensive use of neoadjuvant therapy, macroscopic completeness of total mesorectal excision may not be as valuable a prognosticator as in the past. Larger studies with longer follow-ups are needed to clarify these preliminary findings. See Video Abstract at http://links.lww.com/DCR/C129. LA INTEGRIDAD DE LA ESCISIN MESORRECTAL TODAVA SE CORRELACIONA CON LA RECURRENCIA LOCAL: ANTECEDENTES:La escisión total desl mesorrecto es el estándar de oro para el tratamiento del cáncer de recto. Varios estudios han demostrado una estrecha correlación entre el pronóstico de los pacientes con cáncer de recto y la integridad espécimen mesorrectal.OBJETIVO:Evaluar la correlación entre la evaluación macroscópica de la escisión mesorrectal y los resultados oncológicos a largo plazo en pacientes con cáncer de recto.DISEÑO:Análisis retrospectivo de una base de datos aprobada por el IRB.ENTORNO CLINICO:El estudio se realizó en un centro de referencia terciario de una sola institución.PACIENTES:Todos los pacientes con cáncer de recto operados entre 3/2016-10/2019. Los pacientes se clasificaron en 3 grupos, según la calidad del espécimen mesorrectal: completo, casi completo e incompleto. Solo se incluyeron pacientes con seguimiento >2 años y sin signos de enfermedad a distancia preoperatoria.PRINCIPALES MEDIDAS DE RESULTADO:Identificar la relación entre la escisión mesorrectal total y las tasas de recurrencia local y a distancia en pacientes con cáncer de recto.RESULTADOS:Se incluyeron 124 pacientes (35,5% mujeres) con una edad media de 58,1 años (DE 12) y un índice de masa corporal medio de 26,4 (DE 4,59). Se administró quimiorradiación neoadyuvante al 71% de los pacientes, mientras que el 13,7% recibió terapia neoadyuvante total. Se realizaron procedimientos de restauración en 107 pacientes (86,3%), mientras que 17 pacientes (13,7%) se sometieron a resección abdominoperineal. La mayoría (87,09%) de los especímenes de escisión mesorrectal fueron completas o casi completas. Las tasas de recurrencia local fueron 1/16 (6,3%) en el grupo incompleto y 8/108 (7,4%) en el grupo completo/casi completo ( p = 0,86). Se produjeron metástasis a distancia metacrónicas en 6 pacientes (37,5%) en el grupo incompleto y 24 (22,2%) en el grupo completo/casi completo ( p = 0,18). Por lo tanto, la calidad del espécimen no pareció afectar la supervivencia libre de enfermedad.LIMITACIONES:Estudio retrospectivo de un solo centro con pequeño número de casos y seguimiento relativamente corto.CONCLUSIÓN:En la era de un enfoque multidisciplinario y el uso extensivo de la terapia neoadyuvante, la integridad macroscópica de la escisión total del mesorrecto, puede no ser un pronóstico tan valioso como en el pasado. Se necesitan estudios más amplios con períodos de seguimiento más prolongados para aclarar estos hallazgos preliminares. Consulte Video Resumen en http://links.lww.com/DCR/C129 . (Traducción-Dr. Fidel Ruiz Healy ).

Intracorporeal Vessel Ligation in Laparoscopic Right Colectomy for Cancer is Associated with Increased Lymph Node Yield.

BACKGROUND: This study aimed to compare intra- and extracorporeal division of the vascular pedicle in laparoscopic right colectomy regarding pathological outcomes, short-term morbidity, and local recurrence and distant metastases. METHODS: Retrospective analysis of an IRB-approved database of all patients who underwent laparoscopic right colectomy for cancer between 01/2011 and 08/2021. Main outcome measures were number of harvested lymph nodes, length of resected colon, R1 rate, positive lymph node ratio, short-term post-operative morbidity, local recurrence, and distant metastases. RESULTS: Two-hundred seventy-one consecutive patients (136 males) patients underwent laparoscopic right hemicolectomy for cancer during the study period. Vessel ligation was intracorporeal in 171 (63%) and extracorporeal in 100 patients (37%); groups had similar baseline characteristics except for extent of resection as extended right hemicolectomy was significantly more often performed in the intracorporeal group. When the two groups were matched for the extent of resection (standard versus extended right hemicolectomy), the mean number of harvested lymph nodes (28.61 ± 12.04 versus 25.37 ± 10.06, p = 0.04) and median length of the resected colon [26.00 (IQR: 21.00, 32.00) versus 23.00 (IQR: 19.00, 27.00) cm, p = 0.003] were significantly higher in the intracorporeal than in the extracorporeal group. The intracorporeal group required a significantly longer operative time than did the extracorporeal group (168.94 ± 57.9 vs. 139.7 ± 41.3 mins, p = 0.001). No significant differences were noted between the groups in terms of ileus, hemorrhage, surgical site infection, re-operation rates, recurrence, or distant metastases. CONCLUSION: Intracorporeal vessel ligation in laparoscopic right hemicolectomy was associated with increased lymph node yield and longer specimens, although requiring longer operative times. Postoperative clinical outcomes were similar to outcomes in the extracorporeal ligation group.

Prevention and Treatment of Nonfreezing Cold Injuries and Warm Water Immersion Tissue Injuries: Supplement to Wilderness Medical Society Clinical Practice Guidelines for the Prevention and Treatment of Frostbite.

We convened an expert panel to develop evidence-based guidelines for the evaluation, treatment, and prevention of nonfreezing cold injuries (NFCIs; trench foot and immersion foot) and warm water immersion injuries (warm water immersion foot and tropical immersion foot) in prehospital and hospital settings. The panel graded the recommendations based on the quality of supporting evidence and the balance between benefits and risks/burdens according to the criteria published by the American College of Chest Physicians. Treatment is more difficult with NFCIs than with warm water immersion injuries. In contrast to warm water immersion injuries that usually resolve without sequelae, NFCIs may cause prolonged debilitating symptoms, including neuropathic pain and cold sensitivity.

Interleukin-18 and cytotoxic impairment are independent and synergistic causes of murine virus-induced hyperinflammation.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes typically associated with underlying hematologic and rheumatic diseases, respectively. Familial HLH is associated with genetic cytotoxic impairment and thereby to excessive antigen presentation. Extreme elevation of serum interleukin-18 (IL-18) has been observed specifically in patients with MAS, making it a promising therapeutic target, but how IL-18 promotes hyperinflammation remains unknown. In an adjuvant-induced MAS model, excess IL-18 promoted immunopathology, whereas perforin deficiency had no effect. To determine the effects of excess IL-18 on virus-induced immunopathology, we infected Il18-transgenic (Il18tg) mice with lymphocytic choriomeningitis virus (LCMV; strain Armstrong). LCMV infection is self-limited in wild-type mice, but Prf1-/- mice develop prolonged viremia and fatal HLH. LCMV-infected Il18-transgenic (Il18tg) mice developed cachexia and hyperinflammation comparable to Prf1-/- mice, albeit with minimal mortality. Like Prf1-/- mice, immunopathology was largely rescued by CD8 depletion or interferon-γ (IFNg) blockade. Unlike Prf1-/- mice, they showed normal target cell killing and normal clearance of viral RNA and antigens. Rather than impairing cytotoxicity, excess IL-18 acted on T lymphocytes to amplify their inflammatory responses. Surprisingly, combined perforin deficiency and transgenic IL-18 production caused spontaneous hyperinflammation specifically characterized by CD8 T-cell expansion and improved by IFNg blockade. Even Il18tg;Prf1-haplosufficient mice demonstrated hyperinflammatory features. Thus, excess IL-18 promotes hyperinflammation via an autoinflammatory mechanism distinct from, and synergistic with, cytotoxic impairment. These data establish IL-18 as a potent, independent, and modifiable driver of life-threatening innate and adaptive hyperinflammation and support the rationale for an IL-18-driven subclass of hyperinflammation.

Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains that allows consideration of their dynamic disorder, and used it to analyze a set of D-motif peptide-mitogen-activated protein kinase (MAPK) associations to validate the approach and define unknown binding structures. LiF-MS peptide ligands carry a short-lived, indiscriminately reactive cleavable crosslinker that marks contacts close to ligand binding sites with high specificity. Each marked amino acid provides an independent constraint for a set of directed peptide-protein docking simulations, which are analyzed by agglomerative hierarchical clustering. We found that LiF-MS provides accurate ab initio identification of ligand binding surfaces and a view of potential binding ensembles of a set of D-motif peptide-MAPK associations. Our analysis provides an MKK4-JNK1 structural model, which has thus far been crystallographically unattainable, a potential alternate binding mode for part of the NFAT4-JNK interaction, and evidence of bidirectional association of MKK4 peptide with ERK2. Overall, we find that LiF-MS is an effective noncrystallographic way to understand how short linear motifs associate with specific sites on folded protein domains at the level of individual amino acids.

Ndr/Lats Kinases Bind Specific Mob-Family Coactivators through a Conserved and Modular Interface.

Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and evolutionarily conserved components of "Hippo" signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal regulatory (NTR) region that binds a specific Mob cofactor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the significance of the association of Mob protein with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob cofactors, we determined crystal structures of Saccharomyces cerevisiae Cbk1NTR-Mob2 and Dbf2NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed a significant improvement in the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/LatsNTR-Mob interface provides a distinctive kinase regulation mechanism, in which the Mob cofactor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM), which is involved in allosteric regulation. The Mob-organized NTR appears to mediate association of the HM with an allosteric site on the N-terminal kinase lobe. We also found that Cbk1 and Dbf2 associated specifically with Mob2 and Mob1, respectively. Alteration of residues in the Cbk1 NTR allows association of the noncognate Mob cofactor, indicating that cofactor specificity is restricted by discrete sites rather than being broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/LatsNTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.

Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

Quantitative Human Immunodeficiency Virus (HIV)-1 Antibodies Correlate With Plasma HIV-1 RNA and Cell-associated DNA Levels in Children on Antiretroviral Therapy.

BACKGROUND: This study measured serial plasma human immunodeficiency virus (HIV)-1-specific antibody (Ab) levels in children who initiated antiretroviral therapy (ART) prior to 2 years of age, and evaluated their relationship to peripheral blood HIV-1 RNA and DNA levels. METHODS: We studied 46 HIV-1-infected children, stratified by age at ART initiation (<3 mo, early therapy [ET]; >3 mo-2 years, late therapy [LT]) and by virologic response (R) or non-response (NR), before and up to 4 years following ART. We studied 20 HIV-1-uninfected children born to HIV-1-infected mothers (seroreverters [SR]) as controls. Plasma immunoglobulin G (IgG) Ab levels directed against HIV-1 envelope (gp160, gp41), gag (capsid, p24; matrix, p17), reverse transcriptase (p66/51), and integrase (p31) were serially measured using quantitative enzyme-linked immunosorbent assays. HIV-1 Ab rates of decline were estimated over the first 15 months of the study. RESULTS: The HIV-1 Ab rates of decline in the ET-R group were similar to those in the SR group for all Ab specificities, except for p17 (P = .01). Ab decline rates in the LT-R group and the NR group were significantly slower than in the SR group for all tested Ab specificities. After 1 year of age, Ab levels to p31 and p17 were significantly associated with HIV-1 RNA levels (P < .001); Ab levels to gp160 (P < .001) and gp41 (P < .001) were significantly associated with cell-associated HIV-1 DNA levels. CONCLUSIONS: Quantitative HIV-1-specific Ab levels may be useful for screening children on ART for viral suppression or for residual, cell-associated HIV-1 DNA levels. CLINICAL TRIALS REGISTRATION: NCT00000872.

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection.

Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time (P < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence (P < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection.IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection.

A Theoretical Model Describing the Dynamics of Venous Flow in the DIEP Flap.

BACKGROUND: The deep inferior epigastric artery flap is an integral component of autologous breast reconstruction. The technical aspects of performing the flap have been well-established. A prior mathematical model suggested using the largest perforator and concluded that the inclusion of additional perforators may decrease resistance and increase flow, but at the downside of increased tissue trauma. Many complications may result from inadequate venous drainage of the flap and the same mathematical concepts may be applied. We attempt to give a mathematical model, based on the physics of flow and properties of circuits, to explain clinical observations regarding venous drainage of the flap and the complications that may arise. METHODS: We compare the different possible venous drainage systems of a perforator flap to a complex circuit with multiple resistances. Multiple venous perforators will be represented by resistances in parallel, while the deep and superficial drainage systems will be represented by a complex circuit loop. RESULTS: Drainage of the flap may be optimized through the deep drainage system if the venous perforators are of sufficient size. Inclusion of additional perforators may decrease resistance and enhance drainage. Salvage procedures may be necessary when the venous perforators are insufficient in size or when there are insufficient connections between the deep and superficial systems. CONCLUSION: A single large sized vessel may provide adequate drainage in most DIEP flaps, while the use of multiple vessels may enhance drainage upon the encounter of smaller vessels. Salvage procedures may be needed to relieve venous congestion as the design of the venous system becomes more complicated.

Factors Affecting the Performance of Trickle Dusters for Preventing Explosive Dust Accumulations in Return Airways.

Correctly applied rock dust can dilute, inert, and mitigate the explosive potential of float coal dust. Trickle dusters are one element of a comprehensive system to help prevent coal dust explosions in underground coal mines. Trickle dusters supply rock dust to inert fine float coal dust in areas where it is commonly deposited, such as the longwall tailgate returns, return airways, pillaring areas, and downwind of belt transfers. Dust deposition studies show that the effectiveness of trickle dusters depends on several key factors. Using multiple orifices, rock dust should be released near the mine roof in the direction of the airflow in order to spread the cloud cross the entry. The rock duster should have a mechanism to break up rock dust agglomerates as they leave the rock duster. The particle size distribution of the limestone rock dust and its airborne concentration should be proportional to the airborne size distribution and concentration of coal dust passing by the trickle duster. Specifically, rock dusts having a greater proportion of <74 µm material are more effective at minimizing downwind zones of explosible mixtures than mostly larger particles. In our testing, rock dusts having more than 95% of <74 µm sized particles were adequately dispersed by trickle dusters. Based on our results, the mass rate of rock dust discharge from the trickle duster should exceed the rate of float coal production by at least a factor of four in order to minimize accumulations of explosible dusts.

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis.

The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. IMPORTANCE: Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection of EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains, which may poorly reflect primary viral envelope diversity. We report some of the first primary gp350 sequences, noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development.

The Structure of an NDR/LATS Kinase-Mob Complex Reveals a Novel Kinase-Coactivator System and Substrate Docking Mechanism.

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. "Hippo" pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with "Mob" coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1-Mob2, to our knowledge the first of an NDR/LATS kinase-Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1's regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.

A Gene Expression Signature That Correlates with CD8+ T Cell Expansion in Acute EBV Infection.

Virus-specific CD8(+) T cells expand dramatically during acute EBV infection, and their persistence is important for lifelong control of EBV-related disease. To better define the generation and maintenance of these effective CD8(+) T cell responses, we used microarrays to characterize gene expression in total and EBV-specific CD8(+) T cells isolated from the peripheral blood of 10 individuals followed from acute infectious mononucleosis (AIM) into convalescence (CONV). In total CD8(+) T cells, differential expression of genes in AIM and CONV was most pronounced among those encoding proteins important in T cell activation/differentiation, cell division/metabolism, chemokines/cytokines and receptors, signaling and transcription factors (TF), immune effector functions, and negative regulators. Within these categories, we identified 28 genes that correlated with CD8(+) T cell expansion in response to an acute EBV infection. In EBV-specific CD8(+) T cells, we identified 33 genes that were differentially expressed in AIM and CONV. Two important TF, T-bet and eomesodermin, were upregulated and maintained at similar levels in both AIM and CONV; in contrast, protein expression declined from AIM to CONV. Expression of these TF varied among cells with different epitope specificities. Collectively, gene and protein expression patterns suggest that a large proportion, if not a majority of CD8(+) T cells in AIM are virus specific, activated, dividing, and primed to exert effector activities. High expression of T-bet and eomesodermin may help to maintain effector mechanisms in activated cells and to enable proliferation and transition to earlier differentiation states in CONV.

Design and development of a dust dispersion chamber to quantify the dispersibility of rock dust.

Dispersible rock dust must be applied to the surfaces of entries in underground coal mines in order to inert the coal dust entrained or made airborne during an explosion and prevent propagating explosions. 30 CFR. 75.2 states that "… [rock dust particles] when wetted and dried will not cohere to form a cake which will not be dispersed into separate particles by a light blast of air …" However, a proper definition or quantification of "light blast of air" is not provided. The National Institute for Occupational Safety and Health (NIOSH) has, consequently, designed a dust dispersion chamber to conduct quantitative laboratory-scale dispersibility experiments as a screening tool for candidate rock dusts. A reproducible pulse of air is injected into the chamber and across a shallow tray of rock dust. The dust dispersed and carried downwind is monitored. The mass loss of the dust tray and the airborne dust measurements determine the relative dispersibility of the dust with respect to a Reference rock dust. This report describes the design and the methodology to evaluate the relative dispersibility of rock dusts with and without anti-caking agents. Further, the results of this study indicate that the dispersibility of rock dusts varies with particle size, type of anti-caking agent used, and with the untapped bulk density. Untreated rock dusts, when wetted and dried forming a cake that was much less dispersible than the reference rock dust used in supporting the 80% total incombustible content rule.

TULIPs: tunable, light-controlled interacting protein tags for cell biology.

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

Epstein-Barr virus latent membrane protein 1 genetic variability in peripheral blood B cells and oropharyngeal fluids.

UNLABELLED: We report the diversity of latent membrane protein 1 (LMP1) gene founder sequences and the level of Epstein-Barr virus (EBV) genome variability over time and across anatomic compartments by using virus genomes amplified directly from oropharyngeal wash specimens and peripheral blood B cells during acute infection and convalescence. The intrahost nucleotide variability of the founder virus was 0.02% across the region sequences, and diversity increased significantly over time in the oropharyngeal compartment (P = 0.004). The LMP1 region showing the greatest level of variability in both compartments, and over time, was concentrated within the functional carboxyl-terminal activating regions 2 and 3 (CTAR2 and CTAR3). Interestingly, a deletion in a proline-rich repeat region (amino acids 274 to 289) of EBV commonly reported in EBV sequenced from cancer specimens was not observed in acute infectious mononucleosis (AIM) patients. Taken together, these data highlight the diversity in circulating EBV genomes and its potential importance in disease pathogenesis and vaccine design. IMPORTANCE: This study is among the first to leverage an improved high-throughput deep-sequencing methodology to investigate directly from patient samples the degree of diversity in Epstein-Barr virus (EBV) populations and the extent to which viral genome diversity develops over time in the infected host. Significant variability of circulating EBV latent membrane protein 1 (LMP1) gene sequences was observed between cellular and oral wash samples, and this variability increased over time in oral wash samples. The significance of EBV genetic diversity in transmission and disease pathogenesis are discussed.

Detecting functional divergence after gene duplication through evolutionary changes in posttranslational regulatory sequences.

Gene duplication is an important evolutionary mechanism that can result in functional divergence in paralogs due to neo-functionalization or sub-functionalization. Consistent with functional divergence after gene duplication, recent studies have shown accelerated evolution in retained paralogs. However, little is known in general about the impact of this accelerated evolution on the molecular functions of retained paralogs. For example, do new functions typically involve changes in enzymatic activities, or changes in protein regulation? Here we study the evolution of posttranslational regulation by examining the evolution of important regulatory sequences (short linear motifs) in retained duplicates created by the whole-genome duplication in budding yeast. To do so, we identified short linear motifs whose evolutionary constraint has relaxed after gene duplication with a likelihood-ratio test that can account for heterogeneity in the evolutionary process by using a non-central chi-squared null distribution. We find that short linear motifs are more likely to show changes in evolutionary constraints in retained duplicates compared to single-copy genes. We examine changes in constraints on known regulatory sequences and show that for the Rck1/Rck2, Fkh1/Fkh2, Ace2/Swi5 paralogs, they are associated with previously characterized differences in posttranslational regulation. Finally, we experimentally confirm our prediction that for the Ace2/Swi5 paralogs, Cbk1 regulated localization was lost along the lineage leading to SWI5 after gene duplication. Our analysis suggests that changes in posttranslational regulation mediated by short regulatory motifs systematically contribute to functional divergence after gene duplication.