Mechanism of mycobacterial ATP synthase inhibition by squaramides and second generation diarylquinolines.

Mycobacteria, such as Mycobacterium tuberculosis, depend on the activity of adenosine triphosphate (ATP) synthase for growth. The diarylquinoline bedaquiline (BDQ), a mycobacterial ATP synthase inhibitor, is an important medication for treatment of drug-resistant tuberculosis but suffers from off-target effects and is susceptible to resistance mutations. Consequently, both new and improved mycobacterial ATP synthase inhibitors are needed. We used electron cryomicroscopy and biochemical assays to study the interaction of Mycobacterium smegmatis ATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f. The aryl groups of TBAJ-876 improve binding compared with BDQ, while SQ31f, which blocks ATP synthesis ~10 times more potently than ATP hydrolysis, binds a previously unknown site in the enzyme's proton-conducting channel. Remarkably, BDQ, TBAJ-876, and SQ31f all induce similar conformational changes in ATP synthase, suggesting that the resulting conformation is particularly suited for drug binding. Further, high concentrations of the diarylquinolines uncouple the transmembrane proton motive force while for SQ31f they do not, which may explain why high concentrations of diarylquinolines, but not SQ31f, have been reported to kill mycobacteria.

Nα-Aroyl-N-Aryl-Phenylalanine Amides: A Promising Class of Antimycobacterial Agents Targeting the RNA Polymerase.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of death from a bacterium in the world. The global prevalence of clinically relevant infections with opportunistically pathogenic non-tuberculous mycobacteria (NTM) has also been on the rise. Pharmacological treatment of both TB and NTM infections usually requires prolonged regimens of drug combinations, and is often challenging because of developed or inherent resistance to common antibiotic drugs. Medicinal chemistry efforts are thus needed to improve treatment options and therapeutic outcomes. Nα-aroyl-N-aryl-phenylalanine amides (AAPs) have been identified as potent antimycobacterial agents that target the RNA polymerase with a low probability of cross resistance to rifamycins, the clinically most important class of antibiotics known to inhibit the bacterial RNA polymerase. In this review, we describe recent developments in the field of AAPs, including synthesis, structural characterization, in vitro microbiological profiling, structure-activity relationships, physicochemical properties, pharmacokinetics and early cytotoxicity assessment.

The ΔCysK2 mutant of Mycobacterium tuberculosis is sensitive to vancomycin associated with changes in cell wall phospholipid profile.

Cysteine plays a versatile role in cellular physiology and has previously been shown to be instrumental to Mycobacterium tuberculosis (M.tb) pathophysiology. In this study, we have generated mutants deficient in CysK2 and CysH, the key Cysteine, biosynthetic enzymes. In contrast to the ΔcysH mutant, the ΔcysK2 mutant is not an auxotroph and as such not essential for cysteine biosynthesis. Interestingly, the ΔcysK2 mutant shows increased sensitivity to cumene hydroperoxide, vitamin C, diamide, rifampicin and Vancomycin and shows alterations in phospholipid profile of Mtb cell wall. Our findings suggest that alteration in phospholipids content of M.tb cell wall by CysK2 may form a mode of defence against selected antibiotics and oxidative stress.

Variability of biomarkers used for the classification of metabolic syndrome: A repeated measurements study.

BACKGROUND AND AIMS: The definition of the metabolic syndrome consists of five components. The underlying measurements are subject to intra-individual variability. This repeated measurements study investigated the impact of intra-individual measurement variability on the stability of the diagnosis of metabolic syndrome over 12 months. METHODS AND RESULTS: Twenty-five employees of the University Medicine Greifswald aged 22-70 years were examined once a month over one year. Examinations included blood sampling and anthropometric and blood pressure measurements. Laboratory measurements included glucose, cholesterol (high-density lipoprotein [HDL], and low-density lipoprotein [LDL]), and triglycerides. The metabolic syndrome was defined according to the International Diabetes Federation modified for non-fasting blood samples. Variations in continuous metabolic markers were assessed using coefficients of variation (CV) and intra-class correlation coefficients (ICC). Overall eight participants (32%) were categorized at least once within 12 months as having a metabolic syndrome; in none of those metabolic syndrome was found consistently over the study follow-ups. The Cohen's Kappa for metabolic syndrome was 0.57. CV was highest for triglycerides (27.5%) followed by glucose (10.1%), LDL- (9.5%), and HDL-cholesterol (8.6%). ICC's were lowest for glucose (0.51), triglycerides (0.65), systolic (0.68), and diastolic blood pressure (0.69). CONCLUSION: We showed that the measurement of biomarkers defining the metabolic syndrome is a time-varying condition with implications for the concept of the metabolic syndrome. To account for this uncertainty in prevalence studies we propose to identify uncertain cases according to the current definition of the metabolic syndrome. For analysing associations we recommend to apply probabilistic sensitivity analyses.

Towards the achievement of universal health coverage in the Democratic Republic of Congo: does the Country walk its talk?

In 2009, the Democratic Republic of Congo (DRC) started its journey towards achieving Universal Health Coverage (UHC). This study examines the evolution of financial risk protection and health outcomes indicators in the context of the commitment of DRC to UHC. To measure the effects of such a commitment on financial risk protection and health outcomes indicators, we analyse whether changes have occurred over the last two decades and, if applicable, when these changes happened. Using five variables as indicators for the measurement of the financial risk protection component, there as well retained three indicators to measure health outcomes. To identify time-related effects, we applied the parametric approach of breakpoint regression to detect whether the UHC journey has brought change and when exactly the change has occurred.Although there is a slight improvement in the financial risk protection indicators, we found that the adopted strategies have fostered access to healthcare for the wealthiest quantile of the population while neglecting the majority of the poorest. The government did not thrive persistently over the past decade to meet its commitment to allocate adequate funds to health expenditures. In addition, the support from donors appears to be unstable, unpredictable and unsustainable. We found a slight improvement in health outcomes attributable to direct investment in building health centres by the private sector and international organizations. Overall, our findings reveal that the prevention of catastrophic health expenditure is still not sufficiently prioritized by the country, and mostly for the majority of the poorest. Therefore, our work suggests that DRC's UHC journey has slightly contributed to improve the financial risk protection and health outcomes indicators but much effort should be undertaken.

Which factors are associated with bone marrow oedema suspicious of axial spondyloarthritis as detected by MRI in the sacroiliac joints and the spine in the general population?

OBJECTIVE: Identify factors associated with presence and extension of spinal and sacroiliac joints (SIJ)-MRI lesions suggestive of axial spondyloarthritis (axSpA) in a population-based cohort (Study of Health in Pomerania) aged <45 years. METHODS: Spinal (sagittal T1/T2) and SIJ (semicoronal STIR sequences) MRIs were evaluated by two trained blinded readers. The presence (yes/no) and extension (Berlin MRI Score) of bone marrow oedema (BME) were captured. Degenerative spinal lesions were excluded and discrepancies resolved by consensus. Cross-sectional associations between clinical factors and presence/extension of BME were analysed by logistic/negative binomial regression. Record linkage of claims data was applied to identify participants with axSpA. RESULTS: MRIs of 793 volunteers were evaluated. The presence of SIJ-BME (odds ratio) was strongly associated delivery during the last year (4.47, 1.49-13.41). For SIJ-BME extension, associations (incidence rate ratios, 95% CI) were found for delivery ((during last year) 4.52, 1.48-13.84), human leucocyte antigen (HLA)-B27+ (2.32, 1.30-4.14), body mass index (25-30 vs <25 kg/m²; 1.86 (1.19-2.89)) and back pain ((last 3 months) 1.55, 1.04-2.31), while for spinal BME, associations were found for age per decade (1.46, 1.13-1.90) and physically demanding work (1.46, 1.06-2.00). Record linkage was available for 694 (87.5%) participants and 9/694 (1.3%) had a record of axSpA (ICD M45.09). CONCLUSION: These population-based data support the hypothesis of mechanic strain contributing to BME in the general population aged <45 years and the role of HLA-B27+ as a severity rather than a susceptibility factor for SIJ-BME.

Racemization-free synthesis of Nα-2-thiophenoyl-phenylalanine-2-morpholinoanilide enantiomers and their antimycobacterial activity.

Nα-2-thiophenoyl-D-phenylalanine-2-morpholinoanilide (MMV688845, IUPAC: N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide) from the Pathogen Box® library (Medicines for Malaria Ventures, MMV) is a promising lead compound for antimycobacterial drug development. Two straightforward synthetic routes to the title compound starting from phenylalanine or its Boc-protected derivative are reported. Employing Boc-phenylalanine as starting material and the T3P® and PyBOP® amide coupling reagents enables racemization-free synthesis, avoiding the need for subsequent separation of the enantiomers. The crystal structure of the racemic counterpart gives insight into the molecular structure and hydrogen bonding interactions in the solid state. The R-enantiomer of the title compound (derived from D-phenylalanine) exhibits activity against non-pathogenic and pathogenic mycobacterial strains, whereas the S-enantiomer is inactive. Neither of the enantiomers and the racemate of the title compound shows cytotoxicity against various mammalian cells.

Comparative analysis of a geometric and an adhesive righting strategy against toppling in inclined hexapedal locomotion.

Animals are known to exhibit different walking behaviors in hilly habitats. For instance, cats, rats, squirrels, tree frogs, desert iguana, stick insects and desert ants were observed to lower their body height when traversing slopes, whereas mound-dwelling iguanas and wood ants tend to maintain constant walking kinematics regardless of the slope. This paper aims to understand and classify these distinct behaviors into two different strategies against toppling for climbing animals by looking into two factors: (i) the torque of the center of gravity (CoG) with respect to the critical tipping axis, and (ii) the torque of the legs, which has the potential to counterbalance the CoG torque. Our comparative locomotion analysis on level locomotion and inclined locomotion exhibited that primarily only one of the proposed two strategies was chosen for each of our sample species, despite the fact that a combined strategy could have reduced the animal's risk of toppling over even more. We found that Cataglyphis desert ants (species Cataglyphis fortis) maintained their upright posture primarily through the adjustment of their CoG torque (geometric strategy), and Formica wood ants (species Formica rufa), controlled their posture primarily by exerting leg torques (adhesive strategy). We further provide hints that the geometric strategy employed by Cataglyphis could increase the risk of slipping on slopes as the leg-impulse substrate angle of Cataglyphis hindlegs was lower than that of Formica hindlegs. In contrast, the adhesion strategy employed by Formica front legs not only decreased the risk of toppling but also explained the steeper leg-impulse substrate angle of Formica hindlegs which should relate to more bending of the tarsal structures and therefore to more microscopic contact points, potentially reducing the risk of hindleg slipping.

Facilitating harmonized data quality assessments. A data quality framework for observational health research data collections with software implementations in R.

BACKGROUND: No standards exist for the handling and reporting of data quality in health research. This work introduces a data quality framework for observational health research data collections with supporting software implementations to facilitate harmonized data quality assessments. METHODS: Developments were guided by the evaluation of an existing data quality framework and literature reviews. Functions for the computation of data quality indicators were written in R. The concept and implementations are illustrated based on data from the population-based Study of Health in Pomerania (SHIP). RESULTS: The data quality framework comprises 34 data quality indicators. These target four aspects of data quality: compliance with pre-specified structural and technical requirements (integrity); presence of data values (completeness); inadmissible or uncertain data values and contradictions (consistency); unexpected distributions and associations (accuracy). R functions calculate data quality metrics based on the provided study data and metadata and R Markdown reports are generated. Guidance on the concept and tools is available through a dedicated website. CONCLUSIONS: The presented data quality framework is the first of its kind for observational health research data collections that links a formal concept to implementations in R. The framework and tools facilitate harmonized data quality assessments in pursue of transparent and reproducible research. Application scenarios comprise data quality monitoring while a study is carried out as well as performing an initial data analysis before starting substantive scientific analyses but the developments are also of relevance beyond research.

In Vivo Antimalarial Activity of Leaf Extracts and a Major Compound Isolated from Ranunculus multifidus Forsk.

The leaves of Ranunculus multifidus Forsk. are traditionally used for the treatment of malaria in several African countries. In the present study, 80% methanol (RM-M) and hydrodistilled (RM-H) extracts of fresh leaves from R. multifidus and its major constituent anemonin were tested for their in vivo antimalarial activity against Plasmodium berghei in mice. Anemonin was also tested for its in vitro antimycobacterial activity against Mycobacterium smegmatis and M. abscessus in a microbroth dilution assay, and bacterial growth was analyzed by OD measurement. The isolation of anemonin from RM-H was carried out using preparative thin layer chromatography (PTLC). The chemical structures of anemonin and its hydrolysis product were elucidated using spectroscopic methods (HR-MS; 1D and 2D-NMR). Results of the study revealed that both RM-M and RM-H were active against P. berghei in mice, although the latter demonstrated superior activity (p < 0.001), as compared to the former. At a dose of 35.00 mg/kg/day, RM-H demonstrated a chemosuppression value of 70% in a 4-day suppressive test. In a 4-day suppressive, Rane's and prophylactic antimalarial tests, anemonin showed median effective doses (ED50s) of 2.17, 2.78 and 2.70 µM, respectively. However, anemonin did not inhibit the growth of M. smegmatis and M. abscessus.

Synthesis, structural characterization and antimycobacterial evaluation of several halogenated non-nitro benzothiazinones.

8-Nitro-1,3-benzothiazin-4-ones (BTZs), with BTZ043 and PBTZ169 as the most advanced compounds, represent a new class of potent antitubercular agents, which irreversibly inhibit decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1), an enzyme crucial for cell wall synthesis in the pathogen Mycobacterium tuberculosis. Synthesis, structural characterization and in vitro testing against Mycobacterium aurum DSM 43999 and M. tuberculosis H37Rv of halogenated 2-(4-ethoxycarbonylpiperazin-1-yl)-1,3-benzothiazin-4-ones lacking a nitro group are reported. X-ray crystallography reveals that the structure of the BTZ scaffold can significantly deviate from planarity. In contrast to recent reports, the results of the present study indicate that further investigation of halogenated non-nitro BTZs for antitubercular activity is less than a promising approach.

Frequency of MRI changes suggestive of axial spondyloarthritis in the axial skeleton in a large population-based cohort of individuals aged <45 years.

OBJECTIVE: To investigate the frequency of bone marrow oedema (BME) and fatty lesions (FL) suggestive of axial spondyloarthritis (axSpA) on MRI of the spine and sacroiliac joints (SIJ) in a general population sample. METHODS: As part of a community-based cohort project (Study of Health in Pomerania), volunteers underwent spinal (sagittal T1/T2) and SIJ (semicoronal short tau inversion recovery) MRI examinations. Two calibrated readers evaluated the images to detect BME in SIJ and vertebral corners (VC) and FL in VC suggestive of axSpA using Assessment of SpondyloArthritis international Society definitions. RESULTS: MRIs of 793 volunteers (49.4% males, mean age 37.3±6.3 years, 8.4% human leucocyte antigen-B27+) aged <45 years were evaluated. SIJ BME was seen in 136 (17.2%), VC BME in 218 (27.5%) and FL in 645 (81.4%) volunteers. SIJ BME in ≥1, ≥3 and ≥5 SIJ quadrants was seen in 136 (17.2%), 7 (0.9%) and 1 (0.1%) volunteers, respectively. In VC, BME≥1, ≥3 and ≥5 lesions were seen in 218 (27.5%), 38 (4.8%) and 6 (0.8%) volunteers, respectively, while FL≥1, ≥3 and ≥5 were seen in 645 (81.3%), 351 (44.3%) and 185 (23.3%) volunteers, respectively. Logistic regression analysis showed that BME and FL in VC were related to increasing age: OR 1.33, 95% CI 1.02 to 1.72, and OR 1.73, 95% CI 1.32 to 2.27, per decade increase, respectively. CONCLUSIONS: In this large population-based study, a high frequency of inflammatory and fatty MRI lesions suggestive of axSpA was found, especially in the spine. This indicates a limited value of such MRI findings for diagnosis and classification of axSpA. The increasing frequency with age suggests that mechanical factors could play a role.

The effects of incidental findings from whole-body MRI on the frequency of biopsies and detected malignancies or benign conditions in a general population cohort study.

Magnetic resonance imaging (MRI) yields numerous tumor-related incidental findings (IFs) which may trigger diagnostics such as biopsies. To clarify these effects, we studied how whole-body MRI IF disclosure in a population-based cohort affected biopsy frequency and the detection of malignancies. Laboratory disclosures were also assessed. Data from 6753 participants in the Study of Health in Pomerania (SHIP) examined between 2008 and 2012 were utilized. All underwent laboratory examinations and 3371 (49.9%) a whole-body MRI. Electronic biopsy reports from 2002 to 2017 were linked to participants and assigned to outcome categories. Biopsy frequency 2 years pre- and post-SHIP was investigated using generalized estimating equations with a negative-binomial distribution. Overall 8208 IFs (laboratory findings outside reference limits: 6839; MRI: 1369) were disclosed to 4707 participants; 2271 biopsy reports belonged to 1200 participants (17.8%). Of these, 938 biopsies occurred pre-SHIP; 1333 post-SHIP (event rate/100 observation years = 6.9 [95% CI 6.5; 7.4]; 9.9 [9.3; 10.4]). Age, cancer history, recent hospitalization, female sex, and IF disclosure were associated with higher biopsy rates. Nonmalignant biopsy results increased more in participants with disclosures (post-/pre-SHIP rate ratio 1.39 [95% CI 1.22; 1.58]) than without (1.09 [95% CI 0.85; 1.38]). Malignant biopsy results were more frequent post-SHIP (rate ratio 1.74 [95% CI 1.27; 2.42]). Biopsies increased after participation in a population-based cohort study with MRI and laboratory IF disclosure. Most biopsies resulted in no findings and few malignancies were diagnosed, indicating potential overtesting and overdiagnosis. A more restrictive policy regarding IF disclosure from research findings is required.

[Frequencies of musculoskeletal symptoms and disorders in the population-based German National Cohort (GNC)]. / Häufigkeiten muskuloskelettaler Symptome und Erkrankungen in der bevölkerungsbezogenen NAKO Gesundheitsstudie.

BACKGROUND: Musculoskeletal diseases and symptoms are very common in the general population. They lead to high healthcare costs and pose a significant burden to the national economy. OBJECTIVES: Based on data from the population-based German National Cohort (GNC), frequencies of musculoskeletal symptoms and diseases are reported, including back pain, osteoporosis, osteoarthritis, and arthritis. MATERIALS AND METHODS: Data were collected from March 2014 to March 2017 in adults aged 20-75 years during the first half of the baseline survey of the GNC. The sample comprised 101,779 interviewed subjects, including 9370 subjects who underwent clinical musculoskeletal examinations. The interview included questions about specific musculoskeletal disorders. A clinical examination of the hand provided information about palpable swollen joints and pressure-sensitive joints. Resting pain of the knees and hips was also assessed by a clinical examination. Frequencies were standardized to the German standard population of the year 2011. RESULTS: Having ever been diagnosed with recurrent back pain (22.5%) or osteoarthritis (20.6%) were the most common complaints reported in the interview; osteoporosis (2.9%) and rheumatoid arthritis (1.9%) were stated more seldom. According to the hand examination, 6.0% of all participants experienced pain in at least one finger joint. Resting pain was present in at least one knee among 8.2% and in at least one hip among 5.1% of the participants as assessed during the clinical examination. Women were more likely to report musculoskeletal disorders and symptoms than men. The proportion of adults affected by musculoskeletal diseases increased strongly with age. CONCLUSION: Musculoskeletal disorders and symptoms occur frequently. The burden of complaints and diagnoses is comparable to previous population-based surveys.

THP-1 and Dictyostelium Infection Models for Screening and Characterization of Anti-Mycobacterium abscessus Hit Compounds.

!!NCR1!! presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. M. abscessus is able to grow intracellularly in human macrophages, suggesting that intracellular models can facilitate drug discovery. Thus, we have developed two host cell models: human macrophages for use in a new high-content screening method for M. abscessus growth and a Dictyostelium discoideum infection model with the potential to simplify downstream genetic analysis of host cell factors. A screen of 568 antibiotics for activity against intracellular M. abscessus led to the identification of two hit compounds with distinct growth inhibition. A collection of 317 human kinase inhibitors was analyzed, with the results yielding three compounds with an inhibitory effect on mycobacterial growth, strengthening the notion that host-directed therapy can be applied for M. abscessus.

Guidelines and recommendations for ensuring Good Epidemiological Practice (GEP): a guideline developed by the German Society for Epidemiology.

OBJECTIVE: To revise the German guidelines and recommendations for ensuring Good Epidemiological Practice (GEP) that were developed in 1999 by the German Society for Epidemiology (DGEpi), evaluated and revised in 2004, supplemented in 2008, and updated in 2014. METHODS: The executive board of the DGEpi tasked the third revision of the GEP. The revision was arrived as a result of a consensus-building process by a working group of the DGEpi in collaboration with other working groups of the DGEpi and with the German Association for Medical Informatics, Biometry and Epidemiology, the German Society of Social Medicine and Prevention (DGSMP), the German Region of the International Biometric Society (IBS-DR), the German Technology, Methods and Infrastructure for Networked Medical Research (TMF), and the German Network for Health Services Research (DNVF). The GEP also refers to related German Good Practice documents (e.g. Health Reporting, Cartographical Practice in the Healthcare System, Secondary Data Analysis). RESULTS: The working group modified the 11 guidelines (after revision: 1 ethics, 2 research question, 3 study protocol and manual of operations, 4 data protection, 5 sample banks, 6 quality assurance, 7 data storage and documentation, 8 analysis of epidemiological data, 9 contractual framework, 10 interpretation and scientific publication, 11 communication and public health) and modified and supplemented the related recommendations. All participating scientific professional associations adopted the revised GEP. CONCLUSIONS: The revised GEP are addressed to everyone involved in the planning, preparation, execution, analysis, and evaluation of epidemiological research, as well as research institutes and funding bodies.

[Good Practice Data Linkage]. / Gute Praxis Datenlinkage (GPD).

Individual data linkage of different data sources for research purposes is being increasingly used in Germany in recent years. However, generally accepted methodological guidance is missing. The aim of this article is to define such methodological standards for research projects. Another aim is to provide readers with a checklist for critical appraisal of research proposals and articles. Since 2016, an expert panel of members of different German scientific societies have worked together and developed 7 guidelines with a total of 27 practical recommendations. These recommendations include (1) research aims, questions, data sources and resources, (2) infrastructure and data flow, (3) data privacy, (4) ethics, (5) key variables and type of linkage, (6) data validation/quality assurance and (7) long-term use for future research questions. The authors provide a rationale for each recommendation. Future revisions will include any new developments in science and data privacy.

Screening of Preselected Libraries Targeting Mycobacterium abscessus for Drug Discovery.

Mycobacterium abscessus is intrinsically resistant to many antimycobacterial antibiotics, which presents serious problems in therapy. Here, we describe the development of a novel phenotype-based microscopic and computerized imaging drug screening approach. A pilot screen of 568 compounds from two libraries identified 17 hits. Eleven of these compounds are described for the first time as active against M. abscessus The impact of growth media on the activity of these compounds was tested, revealing that cation-adjusted Mueller-Hinton broth (MHII) supports better growth of actively replicating M. abscessus and improves the activity of associated compounds.

Synthesis, antimycobacterial activity and influence on mycobacterial InhA and PknB of 12-membered cyclodepsipeptides.

In recent years, several small natural cyclopeptides and cyclodepsipeptides were reported to have antimycobacterial activity. Following this lead, a synthetic pathway was developed for a small series of 12-membered ring compounds with one amide and two ester bonds (cyclotridepsipeptides). Within the series, the ring system proved to be necessary for growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis in the low micromolar range. Open-chain precursors and analogues were inactive. The compounds modulated autophosphorylation of the mycobacterial protein kinase B (PknB). PknB inhibitors were active at µM concentration against mycobacteria while inducers were inactive. PknB regulates the activity of the mycobacterial reductase InhA, the target of isoniazid. The activity of the series against Mycobacterium bovis BCG InhA overexpressing strains was indistinguishable from that of the parental strain suggesting that they do not inhibit InhA. All substances were not cytotoxic (HeLa > 5 µg/ml) and did not show any significant antiproliferative effect (HUVEC > 5 µg/ml; K-562 > 5 µg/ml). Within the scope of this study, the molecular target of this new type of small cyclodepsipeptide was not identified, but the data suggest interaction with PknB or other kinases may partly cause the activity.