Improvements in sleep problems and their associations with mental health symptoms: A study of children and adolescents participating in a digital mental health intervention.

Objective: A growing number of youth are utilizing digital mental health interventions (DMHIs) for treatment of mental health problems such as anxiety, depression, and ADHD. Although these mental health symptoms are closely related to sleep problems, it is unknown whether nonsleep DMHIs indirectly confer improvements in sleep. Using retrospective data, the current study assesses (1) whether youth sleep problems improve over participation in a nonsleep DMHI, and (2) whether mental health symptom severity and improvement are correlated with sleep problem severity over time. Methods: Sleep problems and mental health symptoms were assessed every 30 days among children (ages 5-12) and adolescents (ages 13-17) participating in a pediatric digital mental health intervention (DMHI; N = 1219). Results: Children and adolescents with elevated sleep problems (39.3%; n = 479) were older (P < .001), more predominantly female (P < .001), and more likely to have elevated anxiety (P < .001), depressive (P < .001) and inattention symptoms (P = .001), as compared to those with nonelevated sleep problems (60.7%; n = 740). From the baseline to last assessment, 77.3% (n = 269) of members with elevated sleep problems exhibited improvements, with sleep problems decreasing significantly over each month in care (P < .001). Members with improvements in anxiety, depressive, and/or ADHD symptoms had larger improvements in sleep over time compared to their peers with no improvement in their mental health symptoms (Months in care*Change type: P < .001 for all). Conclusions: Our results provide preliminary evidence that participation in a pediatric DMHI is associated with improvements in sleep problems, even when youth are not being treated directly for sleep problems. These findings highlight a valuable secondary benefit of participating in mental health care within pediatric DMHIs and warrant further experimental research.

Rates of Trauma Exposure and Posttraumatic Stress in a Pediatric Digital Mental Health Intervention: Retrospective Analysis of Associations With Anxiety and Depressive Symptom Improvement Over Time.

BACKGROUND: More than 2 out of 3 children and adolescents in the United States experience trauma by the age of 16 years. Exposure to trauma in early life is linked to a range of negative mental health outcomes throughout the lifespan, particularly co-occurring symptoms of posttraumatic stress (PTS), anxiety, and depression. There has been an increasing uptake of digital mental health interventions (DMHIs) among youths, particularly for anxiety and depression. However, little is known regarding the incidence of trauma exposure and PTS symptoms among youths participating in DMHIs and whether PTS symptoms impact anxiety and depressive symptom treatment response. Moreover, it is unclear whether participation in a DMHI for anxiety and depressive symptoms is associated with secondary effects on PTS symptoms among trauma-exposed youths. OBJECTIVE: This study aims to use retrospective data from youths participating in a DMHI to (1) characterize rates of trauma, PTS, and comorbid anxiety and depressive symptoms; (2) determine whether trauma exposure and elevated PTS symptoms impact the improvement of comorbid anxiety and depressive symptoms throughout participation in care; and (3) determine whether participation in a non-posttraumatic DMHI is linked to reductions in PTS symptoms. METHODS: This study was conducted using retrospective data from members (children ages 6 to 12 years) involved in a pediatric collaborative care DMHI. Participating caregivers reported their children's trauma exposure. PTS, anxiety, and depressive symptom severity were measured monthly using validated assessments. RESULTS: Among eligible participants (n=966), 30.2% (n=292) reported at least 1 traumatic event. Of those with trauma exposure and elevated symptoms of PTS (n=119), 73% (n=87) exhibited elevated anxiety symptoms and 50% (n=59) exhibited elevated depressive symptoms. Compared to children with no trauma, children with elevated PTS symptoms showed smaller reductions per month in anxiety but not depressive symptoms (anxiety: F2,287=26.11; P<.001). PTS symptoms also decreased significantly throughout care, with 96% (n=79) of participants showing symptom reductions. CONCLUSIONS: This study provides preliminary evidence for the frequency of trauma exposure and comorbid psychiatric symptoms, as well as variations in treatment response between trauma-exposed and nontrauma-exposed youths, among participants in a pediatric collaborative care DMHI. Youths with traumatic experiences may show increased psychiatric comorbidities and slower treatment responses than their peers with no history of trauma. These findings deliver compelling evidence that collaborative care DMHIs may be well-suited to address mental health symptoms in children with a history of trauma while also highlighting the critical need to assess symptoms of PTS in children seeking treatment.

Improvements in Adolescents' Disordered Eating Behaviors in a Collaborative Care Digital Mental Health Intervention: Retrospective Observational Study.

BACKGROUND: Young people today are exhibiting increasing rates of disordered eating behaviors, as well as eating disorders (EDs), alongside other mental and behavioral problems such as anxiety and depression. However, limited access to mental health care means that EDs, disordered eating behaviors, and comorbid mental health problems are often underdiagnosed and undertreated. Digital mental health interventions (DMHIs) offer accessible and scalable alternatives to traditional treatment modalities, but their effectiveness has not been well established among adolescents with EDs and disordered eating behaviors. OBJECTIVE: This study uses data from a collaborative care pediatric DMHI to determine whether participation in a DMHI is associated with a reduction in adolescents' disordered eating behaviors. METHODS: Adolescent members in care with Bend Health Inc completed the SCOFF questionnaire at baseline (before the start of care) and approximately every month during care to assess disordered eating behaviors. They also completed assessments of mental health symptoms at baseline. Member characteristics, mental health symptoms, and disordered eating behaviors of adolescents with elevated SCOFF scores at baseline (before the start of care) were compared to those of adolescents with nonelevated SCOFF scores at baseline. Members participated in web-based coaching or therapy sessions throughout the duration of mental health care. RESULTS: Compared to adolescents with nonelevated SCOFF scores (n=520), adolescents with elevated SCOFF scores (n=169) were predominantly female and exhibited higher rates of elevated anxiety and depressive symptoms. SCOFF scores decreased over time in care with the DMHI for 61.4% (n=70) of adolescents with elevated SCOFF scores, and each additional month of participation was associated with greater improvements in disordered eating behaviors (F1,233=72.82; P<.001). CONCLUSIONS: Our findings offer promising preliminary evidence that participation in mental health care with a collaborative care DMHI may be beneficial in the reduction of disordered eating symptoms in adolescents, including those who are experiencing comorbid anxiety and depressive symptoms.

Exploring the Number of Web-Based Behavioral Health Coaching Sessions Associated With Symptom Improvement in Youth: Observational Retrospective Analysis.

BACKGROUND: Rates of anxiety and depression have been increasing among children and adolescents for the past decade; however, many young people do not receive adequate mental health care. Digital mental health interventions (DMHIs) that include web-based behavioral health coaching are widely accessible and can confer significant improvements in youth anxiety and depressive symptoms. However, more research is necessary to determine the number of web-based coaching sessions that confer clinically significant improvements in anxiety and depressive symptoms in youth. OBJECTIVE: This study uses data from a pediatric DMHI to explore the number of web-based coaching sessions required to confer symptom improvements among children and adolescents with moderate or moderately severe symptoms of anxiety and depression. METHODS: We used retrospective data from a pediatric DMHI that offered web-based behavioral health coaching in tandem with self-guided access to asynchronous chat with practitioners, digital mental health resources, and web-based mental health symptom assessments. Children and adolescents who engaged in 3 or more sessions of exclusive behavioral health coaching for moderate to moderately severe symptoms of anxiety (n=66) and depression (n=59) were included in the analyses. Analyses explored whether participants showed reliable change (a decrease in symptom scores that exceeds a clinically established threshold) and stable reliable change (at least 2 successive assessments of reliable change). Kaplan-Meier survival analyses were performed to determine the median number of coaching sessions when the first reliable change and stable reliable change occurred for anxiety and depressive symptoms. RESULTS: Reliable change in anxiety symptoms was observed after a median of 2 (95% CI 2-3) sessions, and stable reliable change in anxiety symptoms was observed after a median of 6 (95% CI 5-8) sessions. A reliable change in depressive symptoms was observed after a median of 2 (95% CI 1-3) sessions, and a stable reliable change in depressive symptoms was observed after a median of 6 (95% CI 5-7) sessions. Children improved 1-2 sessions earlier than adolescents. CONCLUSIONS: Findings from this study will inform caregivers and youth seeking mental health care by characterizing the typical time frame in which current participants show improvements in symptoms. Moreover, by suggesting that meaningful symptom improvement can occur within a relatively short time frame, these results bolster the growing body of research that indicates web-based behavioral health coaching is an effective form of mental health care for young people.

Satisfaction, Perceived Usefulness, and Therapeutic Alliance as Correlates of Participant Engagement in a Pediatric Digital Mental Health Intervention: Cross-Sectional Questionnaire Study.

BACKGROUND: Although evidence suggests that digital mental health interventions (DMHIs) are effective alternatives to traditional mental health care, participant engagement continues to be an issue, especially for pediatric DMHIs. Extant studies of DMHIs among adults suggest that participants' satisfaction, perceived usefulness, and therapeutic alliance are closely tied to engagement. However, these associations have not been investigated among children and adolescents involved in DMHIs. OBJECTIVE: To address these gaps in extant DMHI research, the purpose of this study was to (1) develop and implement a measure to assess satisfaction, perceived usefulness, and therapeutic alliance among children and adolescents participating in a DMHI and (2) investigate satisfaction, perceived usefulness, and therapeutic alliance as correlates of children's and adolescents' engagement in the DMHI. METHODS: Members (children and adolescents) of a pediatric DMHI who had completed at least one session with a care provider (eg, coach or therapist) were eligible for inclusion in the study. Adolescent members and caregivers of children completed a survey assessing satisfaction with service, perceived usefulness of care, and therapeutic alliance with care team members. RESULTS: This study provides evidence for the reliability and validity of an adolescent- and caregiver-reported user experience assessment in a pediatric DMHI. Moreover, our findings suggest that adolescents' and caregivers' satisfaction and perceived usefulness are salient correlates of youths' engagement with a DMHI. CONCLUSIONS: This study provides valuable preliminary evidence that caregivers' satisfaction and perceived usefulness are salient correlates of youths' engagement with a DMHI. Although further research is required, these findings offer preliminary evidence that caregivers play a critical role in effectively increasing engagement among children and adolescents involved in DMHIs.

Using Digital Measurement-Based Care for the Treatment of Anxiety and Depression in Children and Adolescents: Observational Retrospective Analysis of Bend Health Data.

BACKGROUND: A growing body of evidence supports the efficacy of measurement-based care (MBC) for children and adolescents experiencing mental health concerns, particularly anxiety and depression. In recent years, MBC has increasingly transitioned to web-based spaces in the form of digital mental health interventions (DMHIs), which render high-quality mental health care more accessible nationwide. Although extant research is promising, the emergence of MBC DMHIs means that much is unknown regarding their effectiveness as a treatment for anxiety and depression, particularly among children and adolescents. OBJECTIVE: This study uses preliminary data from children and adolescents participating in an MBC DMHI administered by Bend Health Inc, a mental health care provider that uses a collaborative care model to assess changes in anxiety and depressive symptoms during participation in the MBC DMHI. METHODS: Caregivers of children and adolescents participating in Bend Health Inc for anxiety or depressive symptoms reported measures of their children's symptoms every 30 days throughout the duration of participation in Bend Health Inc. Data from 114 children (age 6-12 years) and adolescents (age 13-17 years) were used for the analyses (anxiety symptom group: n=98, depressive symptom group: n=61). RESULTS: Among children and adolescents participating in care with Bend Health Inc, 73% (72/98) exhibited improvements in anxiety symptoms and 73% (44/61) exhibited improvement in depressive symptoms, as indicated by either a decrease in symptom severity or screening out of completing the complete assessment. Among those with complete assessment data, group-level anxiety symptom T-scores exhibited a moderate decrease of 4.69 points (P=.002) from the first to the last assessment. However, members' depressive symptom T-scores remained largely stable throughout their involvement. CONCLUSIONS: As increasing numbers of young people and families seek DMHIs over traditional mental health treatments due to their accessibility and affordability, this study offers promising early evidence that youth anxiety symptoms decrease during involvement in an MBC DMHI such as Bend Health Inc. However, further analyses with enhanced longitudinal symptom measures are necessary to determine whether depressive symptoms show similar improvements among those involved in Bend Health Inc.

Using Digital Measurement-Based Care to Address Symptoms of Inattention, Hyperactivity, and Opposition in Youth: Retrospective Analysis of Bend Health.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) and associated behavioral disorders are highly prevalent in children and adolescents, yet many of them do not receive the care they need. Digital mental health interventions (DMHIs) may address this need by providing accessible and high-quality care. Given the necessity for high levels of caregiver and primary care practitioner involvement in addressing ADHD symptoms and behavioral problems, collaborative care interventions that adopt a whole-family approach may be particularly well suited to reduce symptoms of inattention, hyperactivity, and opposition in children and adolescents. OBJECTIVE: The purpose of this study is to use member (ie, child and adolescent) data from Bend Health, Inc, a collaborative care DMHI that uses a whole-family approach to address child and adolescent mental health concerns, to (1) determine the effects of a collaborative care DMHI on inattention, hyperactivity, and oppositional symptoms in children and adolescents and (2) assess whether the effects of a collaborative care DMHI vary across ADHD subtypes and demographic factors. METHODS: Caregivers of children and adolescents with elevated symptoms of inattention, hyperactivity, or opposition assessed their children's symptom severity approximately every 30 days while participating in Bend Health, Inc. Data from 107 children and adolescents aged 6-17 years who exhibited clinically elevated symptoms at baseline were used to assess symptom severity across monthly assessments (inattention symptom group: n=91, 85.0%; hyperactivity symptom group: n=48, 44.9%; oppositional symptom group: n=70, 65.4%). The majority of the sample exhibited elevated symptoms of at least 2 symptom types at baseline (n=67, 62.6%). RESULTS: Members received care for up to 5.52 months and attended between 0 and 10 coaching, therapy, or psychiatry sessions through Bend Health, Inc. For those with at least 2 assessments, 71.0% (n=22) showed improvements in inattention symptoms, 60.0% (n=9) showed improvements in hyperactivity symptoms, and 60.0% (n=12) showed improvements in oppositional symptoms. When considering group-level change over time, symptom severity decreased over the course of treatment with Bend Health, Inc, for inattention (average decrease=3.51 points, P=.001) and hyperactivity (average decrease=3.07 points, P=.049) but not for oppositional symptoms (average decrease=0.70 points, P=.26). There was a main effect of the duration of care on symptom severity (P<.001) such that each additional month of care was associated with lower symptom scores. CONCLUSIONS: This study offers promising early evidence that collaborative care DHMIs may facilitate improvements in ADHD symptoms among children and adolescents, addressing the growing need for accessible and high-quality care for behavioral health problems in the United States. However, additional follow-up studies bolstered by larger samples and control groups are necessary to further establish the robustness of these findings.

Evidence Base for Measurement-Based Care in Child and Adolescent Psychiatry.

Measurement-based care (MBC) is the routine collection of data using standardized, validated measures and use of these data to guide treatment. MBC has been implemented throughout medicine to improve patient outcomes, but its use in mental health care remains low. This article summarizes the evidence of MBC's efficacy in adults then reviews the sparser data in youth mental health care. The literature indicates that MBC must be administered immediately before or during every encounter, results must be available to and reviewed by providers immediately, results must be shared with the patient, and results must be used to guide treatment.

Wear and creep behavior of total knee implants undergoing wear testing.

We sought to determine what dimensional changes occurred from wear testing of a total knee implant, as well as any changes within the polyethylene subsurface. Three fixed bearing implants underwent wear simulator testing to 6.1 million cycles. Gravimetric analysis and micro-CT scans were performed pre-test, mid-test, and post-test. Wear volume and surface deviations were greater during 0-3.2 million cycles (91 ± 12mm(3)) than from 3.2 to 6.1 million cycles (52 ± 18mm(3)). Deviations (wear and creep) occurred across all surfaces of the tibial inserts, including the articular surface, backside surface, sides, and locking mechanism. No subsurface changes were found. The micro-CT results were a useful adjunct to gravimetric analysis, defining the dimensional changes that occurred with testing and ruling out subsurface fatigue.

Development of an animal model to investigate optimal laparoscopic trocar site fascial closure.

BACKGROUND: The rate of hernia formation after closure of 10-12 mm laparoscopic trocar sites is grossly under-reported. Using an animal model, we have developed a method to assess trocar site fascial dehiscence and the strength of different methods of fascial closure. MATERIALS AND METHODS: Pigs (n = 9; 17 ± 2.5 lbs) underwent placement of 12 mm Hasson trocars with pneumoperitoneum maintained for 1 h. Three closure techniques (Figure-of-eight; simple interrupted; pulley) were compared with no fascial closure and to native fascia at five randomly allocated abdominal wall midline locations. Necropsy was performed on the fourth postoperative d. Statistical comparisons of tensile strength and breaking strength based on closure type and trocar location were made using ANOVA with Tukey's tests. RESULTS: The mean (SD) force (Newtons) required for fascial disruption varied significantly with closure type [Native Fascia 170 (39), Figure-of-eight 169 (31), Pulley 167 (59), Simple Interrupted 151 (27), No Closure 108 (28)]; P = 0.007. The mean force required for fascial disruption was significantly increased for Native Fascia, Figure-of-eight, and Pulley relative to No Closure (P = 0.013, P = 0.015, P = 0.023, respectively). The mean (SD) force (in Newtons) required for fascial disruption also varied significantly with location of trocar [subxiphoid 181 (43), supraumbilical 151 (23), Umbilical 146 (23), infraumbilical 168 (62), suprapubic 120 (38)]; P = 0.03. The mean force for subxiphoid location was significantly increased relative to the suprapubic location (P = 0.021). CONCLUSIONS: We have developed a novel assessment model that reliably detects differences in fascial integrity after laparoscopic trocar placement and closure. This model will allow for further testing of various trocars and closure techniques, and facilitate hernia prevention strategies.

Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria.

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Baseball and softball sliding injuries: incidence and correlates during one high school league varsity season.

OBJECTIVE: To estimate injury rates associated with sliding in high school baseball and softball. DESIGN: Prospective cohort study. SETTING: Community high school athletic events. PARTICIPANTS: Ten high school varsity baseball and softball teams over 1 season. ASSESSMENT OF RISK FACTORS: All sliding attempts were recorded during each game and recorded as headfirst, feetfirst, or diveback. Base type, playing surface, and field conditions were also noted. MAIN OUTCOME MEASURES: Injury exposure rates by game exposures and sliding/diveback exposures. RESULTS: Data were collected from 153 baseball games and 166 softball games. A greater proportion of slides were associated with injury in softball than in baseball (42.0 and 4.9 per 1000 slides; P < 0.05). Headfirst slides led to more injuries than feetfirst slides in baseball (16.8 vs 0 per 1000 slides; P < 0.05) but not in softball (55 vs 35 per 1000 slides; P = 0.74). CONCLUSIONS: More powerful studies are required to determine whether efforts to prevent baseball sliding injuries at the high school level should focus on better education in sliding technique or changes in equipment. Softball players are vulnerable to injury when wearing inadequate protective sliding apparel.

Management options for symptomatic stenosis after laparoscopic vertical sleeve gastrectomy in the morbidly obese.

BACKGROUND: This study aimed to determine the incidence, etiology, and management options for symptomatic stenosis (SS) after laparoscopic sleeve gastrectomy (LSG). METHODS: A retrospective study reviewed morbidly obese patients who underwent LSG between October 2008 and December 2010 to identify patients treated for SS. RESULTS: In this study, 230 patients (83% female) with a mean age of 49.5 years and a mean body mass index (BMI) of 43 kg/m(2) underwent LSG. In 3.5% of these patients (100% female; mean age, 42 years; mean BMI, 42.6 kg/m(2)), SS developed. The LSG procedure was performed using a 36-Fr. bougie and tissue-reinforced staplers. Four patients had segmental staple-line imbrication, and seven patients underwent contrast study, with 71.4% demonstrating a fixed narrowing. Endoscopy confirmed short-segment stenoses: seven located at mid-body and one located near the gastroesophageal junction. Endoscopic management was 100% successful. The mean number of dilations was 1.6, and the median balloon size was 15 mm. The mean time from surgery to initial endoscopic intervention was 48.8 days, and the mean time from the first dilation to toleration of a solid diet was 49.6 days. Two patients were referred to our institution after undergoing LSG at another facility. The mean time to the transfer was 28.5 days. The two patients had a mean age of 35 years and a mean BMI of 42.3 kg/m(2). Both patients experienced immediate SS after perioperative complications comprising one staple-line hematoma and one leak. Contrast studies demonstrated minimal passage of contrast through a long-segment stenosis. Both patients underwent multiple endoscopic dilation procedures and endoluminal stenting, ultimately requiring laparoscopic conversion to Roux-en-Y gastric bypass. The mean time from the initial surgery to the surgical revision was 77 days, and the mean time after the first intervention to tolerance of a solid diet was 82 days. CONCLUSION: Symptomatic short-segment stenoses after LSG may be treated successfully with endoscopic balloon dilation. Long-segment stenoses that do not respond to endoscopic techniques may ultimately require conversion to Roux-en-Y gastric bypass.

Phosphorylation of enoyl-acyl carrier protein reductase InhA impacts mycobacterial growth and survival.

InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.

Structure of N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Mycobacterium tuberculosis in a cubic space group.

GlmU is a bifunctional enzyme that catalyzes the final two steps in the biosynthesis of UDP-GlcNAc. Crystals of GlmU from Mycobacterium tuberculosis obtained using ammonium sulfate as a precipitant diffracted poorly (to 3.4 A resolution) and displayed an unusually high solvent content (>80%) with sparse crystal packing that resulted in large solvent channels. With one molecule per asymmetric unit, the monomers from three neighbouring asymmetric units related by the crystal threefold formed a biological trimer. Although this is the first report of the structure of GlmU determined in a cubic crystal form, the trimeric arrangement here is similar to that observed for other GlmU structures determined in hexagonal (H3, H32, P6(3)22) space groups.

The Mycobacterium tuberculosis protein kinase K modulates activation of transcription from the promoter of mycobacterial monooxygenase operon through phosphorylation of the transcriptional regulator VirS.

Mycobacterium tuberculosis encodes for 11 eukaryotic-like serine/threonine protein kinases. Genetic and biochemical studies show that these kinases regulate various cellular processes including cell shape and morphology, glucose and glutamine transport, phagosome-lysosome fusion and the expression, and/or activity of transcription factors. PknK is the largest predicted serine/threonine protein kinase in M. tuberculosis. Here, we have cloned, overexpressed, and purified protein kinase K (PknK) to near homogeneity and show that its ability to phosphorylate proteins is dependent on the invariant lysine (Lys(55)), and on two conserved threonine residues present in its activation loop. Despite being devoid of any apparent transmembrane domain, PknK is localized to the cell wall fraction, suggesting probable anchoring of the kinase to the cell membrane region. The pknK gene is located in the vicinity of the virS gene, which is known to regulate the expression of the mycobacterial monooxygenase (mymA) operon. We report here for the first time that VirS is in fact a substrate of PknK. In addition, four of the proteins encoded by mymA operon are also found to be substrates of PknK. Results show that VirS is a bona fide substrate of PknK in vivo, and PknK-mediated phosphorylation of VirS increases its affinity for mym promoter DNA. Reporter assays reveal that PknK modulates VirS-mediated stimulation of transcription from the mym promoter. These findings suggest that the expression of mymA operon genes is regulated through PknK-mediated phosphorylation of VirS.

PknB-mediated phosphorylation of a novel substrate, N-acetylglucosamine-1-phosphate uridyltransferase, modulates its acetyltransferase activity.

Identifying direct targets of kinases and determining how their activities are regulated are central to understanding how they generate biological responses. Genetic and biochemical studies have shown that Mycobacterium tuberculosis serine/threonine protein kinases PknA and PknB play a role in modulating cell shape and possibly cell division. In this report, we show that the enzyme N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) of M. tuberculosis is a novel substrate of PknB and is phosphorylated on threonine residues. GlmU carries out two important biochemical activities: a C-terminal domain catalyzes the transfer of acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce N-acetylglucosamine-1-phosphate, which is converted into UDP-N-acetylglucosamine by the transfer of uridine 5'-monophosphate (from uridine 5'-triphosphate), a reaction catalyzed by the N-terminal domain. We determined the crystal structures of GlmU in apo form and UDP-N-acetylglucosamine-bound form, and analyzed them to identify threonine residues that may be accessible to PknB. The structure shows a two-domain architecture, with an N-terminal domain having an alpha/beta-like fold and with a C-terminal domain that forms a left-handed parallel beta-helix structure. Kinase assays with PknB using the N- and C-terminal domains of GlmU as substrates illustrated that PknB phosphorylates GlmU in the C-terminal domain. Furthermore, mutational studies reveal one of the five threonines present in region 414-439 to be phosphorylated by PknB. Structural and biochemical analyses have shown the significance of a variable C-terminal tail in regulating acetyltransferase activity. Notably, we demonstrate that although PknB-mediated phosphorylation of GlmU does not affect its uridyltransferase activity, it significantly modulates the acetyltransferase activity. These findings imply a role for PknB in regulating peptidoglycan synthesis by modulating the acetyltransferase activity of GlmU.

Cyanobacterial flora from polluted marine shores.

Citation of cyanobacterial cultures from the shores of south west coast of Gujarat, India and their relationship with sea water quality, influenced by extensive pollutant runoff is reported in this study. Intensity of pollution was evaluated by physico-chemical analysis of water. Higher load of suspended solids (60-1000 mg l(-1)) and nutrients (PO4- P: 1.3-4 micromole l(-1) and NO3- N: 12.5-17.8 micromole l(-1)) were persistent throughout the analysis. Community structure is seen to be influenced by such persistent pollution. Twenty nine cyanobacterial species were isolated belonging to 9 genera of 4 families, with an elevated occurrence of Oscillatoria and Lyngbya species. No heterocystous cyanobacteria were isolated throughout the study.

Cyanobacterial flora from polluted industrial effluents.

Effluents originating from pesticides, agro-chemicals, textile dyes and dyestuffs industries are always associated with high turbidity, colour, nutrient load, and heavy metals, toxic and persistent compounds. But even with such an anthropogenic nature, these effluents contain dynamic cyanobacterial communities. Documentation of cyanobacterial cultures along the water channels of effluents discharged by above mentioned industries along the west coast of India and their relationship with water quality is reported in this study. Intensity of pollution was evaluated by physico-chemical analysis of water. Higher load of solids, carbon and nutrients were found to be persistent throughout the analysis. Sediment and water samples were found to be colored in nature. Cyanobacterial community structure was found to be influenced by the anthropogenic pollution. 40 different cyanobacterial species were recorded from 14 genera of 5 families and an elevated occurrence of Phormidium, Oscillatoria and Chroococcus genera was observed in all the sampling sites.

Partial characterization of extracellular polysaccharides from cyanobacteria.

Four cyanobacterial strains, Cyanothece sp., Oscillatoria sp., Nostoc sp. and Nostoc carneum were studied for physico-chemical characterization of extracellular polysaccharide (EPS) secreted during the controlled growth condition. Hydrolyzed EPSs showed the compositional involvement of four sugar moieties viz. mannose, glucose, xylose and ribose in varying combinations. Infrared spectra of EPSs showed a specific absorbance of O-H stretching at 3448-3400 cm(-1), asymmetrical-symmetrical C-H stretching at 2924 and 2854 cm(-1) and a bending vibration of C-H at 1400-1380 cm(-1). Absorbance at 1259 and 1140 cm(-1) with Cyanothece sp. EPS, indicated the presence of sulfur containing functional group. Thermal gravimetric analysis and differential scanning calorimetric analysis confirmed the polysaccharides thermal stability as high as around 250 degrees C. In the presence of 0.1 M NaCl aqueous solution, the intrinsic viscosity of polysaccharides from Oscillatoria sp. and Nostoc sp. decreased 1.6 fold, whereas, 3-5 fold reduction in intrinsic viscosity was observed with commercially available guar and xanthan gum.