Pro přežití výkyvů vnějších podmínek bakterie vyvinuly stresové adaptační mechanismy, ke kterým patří i stringentní odpověď, která je řízena hladinou nukleotidové signální molekuly (p)ppGpp. Klíčovými regulátory tohoto procesu jsou multifunkční enzymy Rel a RelA/SpoT z rodiny proteinů RSH. Přestože jsou komponenty a funkce stringentní odpovědi u bakterií vysoce konzervované, mohou se u některých bakterií konkrétní molekulární mechanismy regulace značně lišit. Stringentní odpověď a její komponenty ovlivňují celou řadu buněčných pochodů a představují tak potenciální cíle pro vývoj nových typů léčiv proti infekcím způsobených rezistentními bakteriálními patogeny.

To survive fluctuations of the environmental conditions, the bacteria have evolved stress response mechanisms, such as the stringent response, which is controlled by the changes of intracellular signal molecule (p)ppGpp levels. The key regulators of this process are the multifunctional enzymes Rel and RelA/SpoT of the RSH protein family. Components and functions of the stringent response are highly conserved throughout the bacterial domain, yet the particular molecular mechanisms of its regulation can differ strongly between species. Stringent response and its components play a role in the whole spectrum of cellular functions and represent a potential target for development of novel drugs against infections caused by resistant bacterial pathogens.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fyziologický stres * MeSH
• ligasy genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Staphylococcus aureus genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We identify here a pattern in the transcription start sites (+1A or +1G) of sigma(A)-dependent promoters of genes that are up-/downregulated in response to amino acid starvation (stringent response) in Bacillus subtilis. Upregulated promoters initiate mostly with ATP and downregulated promoters with GTP. These promoters appear to be sensitive to changes in initiating nucleoside triphosphate concentrations. During the stringent response in B. subtilis, when ATP and GTP levels change reciprocally, the identity of the +1 position (A or G) of these promoters is a factor important in their regulation. Mutations that change the identity of position +1 (A for G and vice versa) change the response of the promoter to amino acid starvation.

• MeSH
• aminokyseliny metabolismus   MeSH
• Bacillus subtilis genetika   metabolismus   MeSH
• bakteriální proteiny genetika   MeSH
• financování organizované MeSH
• genetická transkripce MeSH
• nukleotidy metabolismus   MeSH
• počátek transkripce MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH

Recent studies in adult lymphoma patients have indicated a correlation between polymorphisms of Fc gamma-receptors (FcγRs, encoded by the respective FCGR genes) and the response to rituximab treatment. In vitro, cells expressing FcγRIIIa-158V mediate antibody-dependent cellular cytotoxicity (ADCC) more efficiently than cells expressing FcγRIIIa-158F. The impact of the FCGR2A-131HR polymorphism is unclear. In this study, the FCGR polymorphisms FCGR3A-158VF and FCGR2A-131HR were analyzed in pediatric patients with mature aggressive B cell non-Hodgkin lymphoma/leukemia (B-NHL). Pediatric patients received a single dose of rituximab monotherapy. Response was evaluated on day 5 followed by standard chemotherapy for B-NHL. Among 105 evaluable patients, a response to rituximab was observed in 21 % of those homozygous for FcγRIIa-131RR (5/24) compared to 48 % of patients who were HH and HR FcγRIIa-131 allele carriers (18/34 and 21/47, respectively; p = 0.044). Among patients with the FCGR3A-158 polymorphism, those homozygous for the FF genotype had a significantly favorable rituximab response rate of 59 % (22/37) compared to 32 % in patients who were FcγRIIIa-158VV and FcγRIIIa-VF allele carriers (2/9 and 20/59, respectively; p = 0.022). A stringent phase II response evaluation of children and adolescents with B-NHL after one dose of rituximab monotherapy showed a significant association between the rituximab response rate and FCGR polymorphisms. These findings support the hypothesis that FCGR polymorphisms represent patient-specific parameters that influence the response to rituximab.

• MeSH
• B-buněčný lymfom krev   farmakoterapie   genetika   MeSH
• dítě MeSH
• frekvence genu MeSH
• genotyp MeSH
• indukce remise MeSH
• jednonukleotidový polymorfismus * MeSH
• L-laktátdehydrogenasa krev   metabolismus   MeSH
• lidé MeSH
• lokální recidiva nádoru MeSH
• mladiství MeSH
• multivariační analýza MeSH
• prognóza MeSH
• protinádorové látky terapeutické užití   MeSH
• receptory IgG genetika   MeSH
• rituximab terapeutické užití   MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

CONTEXT: Achieving minimal residual disease negativity (MRD-) in MM is associated with improved survival. Isatuximab (Isa) plus carfilzomib (K) and dexamethasone (d) is approved for relapsed MM patients after ≥1 prior therapy, based on IKEMA interim analysis (NCT03275285). OBJECTIVE: Report updated, longer-term depth of response (DoR) results from IKEMA, including MRD-status. DESIGN AND PATIENTS: IKEMA, a randomized, open-label, multicenter Phase 3 study, investigates Isa-Kd (n=179) versus Kd (n=123) in relapsed MM patients with 1-3 prior lines of therapy. INTERVENTIONS: IV Isa 10 mg/kg was given QW for 4 weeks, followed by Q2W. Both arms received an approved schedule of K (IV) and d (oral/IV). MAIN OUTCOME MEASURES: This prespecified analysis evaluated PFS (primary endpoint) at 159 events; secondary endpoints were ≥CR (+stringent CR), MRD-, and ≥CR+MRD-rates, as determined by IRC based on central laboratory data and review of local radiology. MRD status was assessed by NGS (sensitivity threshold ≥10-5) in bone marrow aspirates from patients achieving ≥VGPR. HYDRASHIFT Isa immunofixation (IFE) test, removing Isa interference in IFE, was used to update ≥CR rate. Secondary endpoints were compared between treatment arms using Cochran-Mantel-Haenszel test. One-sided descriptive P-values were provided. All randomized patients not reaching MRD- or without MRD assessment were considered MRD+. RESULTS: At cutoff (14-Jan-2022), with a median follow-up of 44 months, deeper responses were observed in Isa-Kd versus Kd, ≥CR rates 44.1% versus 28.5% (OR: 2.09; 95%CI=1.26-3.48; descriptive P=0.0021). For Isa-Kd versus Kd patients, MRD- (10-5) occurred in 33.5% versus 15.4% (OR: 2.78; 95%CI=1.55-4.99; descriptive P=0.0002), with 26.3% versus 12.2% reaching ≥CR+MRD- (10-5; OR: 2.57; 95%CI=1.35-4.88; P=0.0015); MRD- at 10-6 sensitivity occurred in 10.6% versus 3.3%. MRD- versus MRD+ patients (10-5) had longer mPFS (months); Isa-Kd: not calculable ([NC]; 95%CI=NC-NC) versus 21.7 (95%CI=16.4-27.1); Kd: NC (95%CI=29.2-NC) versus 16.2 (95%CI=13.4-19.5). CONCLUSIONS: These results demonstrate clinically meaningful improvement in DoR with Isa-Kd versus Kd. Impressive ≥CR and ≥CR+MRD- (10-5) rates in Isa-Kd versus Kd are the highest reported for proteasome inhibitor-based regimens in relapsed MM. Achieving MRD- led to better outcomes in both arms; Isa-Kd patients had >2-fold higher likelihood of achieving MRD-. Additionally, Isa improved outcomes of MRD+ patients.

• MeSH
• dexamethason škodlivé účinky   MeSH
• humanizované monoklonální protilátky MeSH
• inhibitory proteasomu terapeutické užití   MeSH
• lidé MeSH
• lokální recidiva nádoru farmakoterapie   MeSH
• mnohočetný myelom * MeSH
• oligopeptidy MeSH
• protokoly protinádorové kombinované chemoterapie škodlivé účinky   MeSH
• výsledek terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• randomizované kontrolované studie MeSH

BACKGROUND: Planktonic stationary and exponential cultures of Pseudomonas aeruginosa are highly resistant to killing by bactericidal antimicrobials because of the presence of persisters, cells that are multidrug tolerant and play a key role in the recalcitrance of biofilm infections. AIM: The aim of this study was to investigate the formation of persister cells in P. aeruginosa stationary vs. exponential cultures using different class antimicrobials. METHODS: The susceptibilities of P. aeruginosa PAO1 wild-type and mutant strains to antimicrobials were determined by standard microtiter broth dilution method. In order to determine persister formation, dose- and time-dependent killing experiments were performed with antibiotics. RESULTS: Ceftazidime (Cephalosporin) showed little efficacy against either culture. Stationary-phase cells were more tolerant to imipenem (Carbapenem) than exponential cells, leaving a small fraction of persisters at high imipenem concentration in both populations. Polymyxin B (Polymyxin) appeared to be ineffective at low concentrations against both cell populations. Very high polymyxin B concentration completely eradicated exponential cells and regrowth was seen in a stationary population. Stationary cells were more tolerant to tobramycin (Aminoglycoside) than exponential cells but a higher concentration of tobramycin completely eliminated survivors. Ciprofloxacin (Fluoroquinolone) at a low concentration resulted in killing of both cultures of P. aeruginosa, producing persisters that were invulnerable to killing. CONCLUSIONS: Stationary cells appear to be somewhat more tolerant than exponential cells in all of these assays. We also showed that nutrient deprivation (serine starvation) regulated by stringent and general stress response, contribute to the increased tolerance of P. aeruginosa exponential and stationary planktonic cells via production of persisters.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence účinky léků   MeSH
• biofilmy účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• Pseudomonas aeruginosa účinky léků   MeSH
• serin nedostatek   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Publikační typ
• časopisecké články MeSH

Dendritic cells (DC) are a promising tool for vaccine therapy due to their unique properties as antigen presenting cells and their ability to prime naive T cells. Increasing evidence suggests that maturation stage of DC critically influences the fate of the immune response. Generation of monocyte-derived DC for clinically applicable immunotherapy requires the use of well-defined components and stringent culture conditions. An alternative strategy is to use human autologous serum. However, its constituents are not stable and reflect the inflammatory condition of the donor. In order to investigate whether DC properties are influenced by proteins present in the plasma, we matured human monocyte-derived DC with four main plasma components: fibrinogen, fibronectin, plasminogen or C-reactive protein. These purified proteins were added at various concentrations on day 6 after the initial differentiation induced by IL-4 and GM-CSF. The maturation was assessed by phenotyping of maturation-associated marker (CD83) and co-stimulatory molecule CD86 as well as IL-12 production. Functional properties of DC were assessed by endocytic activity and mixed leukocyte culture. Our results indicate that fibrinogen had DC-maturation effect comparable to poly-I:C, TNF-alpha and PGE(2) as a positive control, but it failed to induce IL-12 production. The other plasma proteins had no effect on DC maturation. CRP at high concentration had rather inhibitory effect on DC induced lymphocyte function. We conclude that none of the tested plasma components and acute phase proteins sufficiently induce fully competent mature DC. This finding is important for the preparation of human DC-based vaccines supplemented by autologous sera.

• MeSH
• buněčná diferenciace účinky léků   MeSH
• CD antigeny analýza   MeSH
• dendritické buňky * imunologie   účinky léků   MeSH
• fibrinogen farmakologie   MeSH
• imunofenotypizace MeSH
• imunoglobuliny analýza   MeSH
• krevní proteiny * farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• melanom krev   MeSH
• membránové glykoproteiny analýza   MeSH
• monocyty * imunologie   účinky léků   MeSH
• test smíšené lymfocytární kultury MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• srovnávací studie MeSH

UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• deoxyguanosin analogy a deriváty   biosyntéza   chemie   MeSH
• dipeptidy biosyntéza   chemie   MeSH
• Enterococcus faecalis účinky léků   enzymologie   genetika   metabolismus   MeSH
• fyziologický stres MeSH
• guanosindifosfát metabolismus   MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintetrafosfát biosyntéza   MeSH
• guanosintrifosfát metabolismus   MeSH
• hořčík MeSH
• ligasy genetika   metabolismus   MeSH
• molekulární struktura MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u bakterií MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• Betacoronavirus MeSH
• COVID-19 MeSH
• epidemický výskyt choroby MeSH
• trasování kontaktů MeSH

• Konspekt
• Veřejné zdraví a hygiena
• NLK Obory
• veřejné zdravotnictví
• infekční lékařství
• NLK Publikační typ
• publikace WHO

Tick-borne encephalitis (TBE) represents one of the most serious arboviral neuro-infections in Europe and northern Asia. As no specific antiviral therapy is available at present, there is an urgent need for efficient drugs to treat patients with TBE virus (TBEV) infection. Using two standardised in vitro assay systems, we evaluated a series of 29 nucleoside derivatives for their ability to inhibit TBEV replication in cell lines of neuronal as well as extraneural origin. The series of tested compounds included 2'-C- or 2'-O-methyl substituted nucleosides, 2'-C-fluoro-2'-C-methyl substituted nucleosides, 3'-O-methyl substituted nucleosides, 3'-deoxynucleosides, derivatives with 4'-C-azido substitution, heterobase modified nucleosides and neplanocins. Our data demonstrate a relatively stringent structure-activity relationship for modifications at the 2', 3', and 4' nucleoside positions. Whereas nucleoside derivatives with the methylation at the C2' position or azido modification at the C4'position exerted a strong TBEV inhibition activity (EC50 from 0.3 to 11.1 μM) and low cytotoxicity in vitro, substitutions of the O2' and O3' positions led to a complete loss of anti-TBEV activity (EC50 > 50 μM). Moreover, some structural modifications of the heterobase moiety resulted in a high increase of cytotoxicity in vitro. High antiviral activity and low cytotoxicity of C2' methylated or C4' azido substituted pharmacophores suggest that such compounds might represent promising candidates for further development of potential therapeutic agents in treating TBEV infection.

• MeSH
• antivirové látky chemie   farmakologie   MeSH
• buněčné linie MeSH
• klíšťová encefalitida farmakoterapie   virologie   MeSH
• molekulární struktura MeSH
• nukleosidy chemie   farmakologie   MeSH
• prasata MeSH
• replikace viru účinky léků   MeSH
• viry klíšťové encefalitidy účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH