Estimation of the appendicular skeletal muscle mass and phase angle cut-off values from a young Turkish reference population using multifrequency bioelectrical impedance analysis.

OBJECTIVE: Population-specific muscle mass cut-off values are recommended for the diagnosis of sarcopenia. In this study, we aimed to determine the appendicular muscle mass index (ASMI) and phase angle (PA) cut-off values for the Turkish population using multi-frequency bioelectrical impedance analysis (mBIA). PATIENTS AND METHODS: A total of 250 healthy volunteers aged 18-40 years were included in the study between September 2020 and December 2021. PA was measured by mBIA, and appendicular skeletal muscle mass (ASM) was calculated by the Sergi formula using the resistance and reactance measurements from mBIA. ASMI was calculated as ASM (kg)/(height in meters)2. Two standard deviations (SD) below the mean values were accepted as cut-off points. RESULTS: 134 women and 116 men were included in the study (26.0±5.6 years). The ASMI cut-offs for men and women were 5.86 and 4.36 kg/m2, respectively. The PA cut-offs were 5.66° in men and 4.38° in women. CONCLUSIONS: The present study reported the ASMI and PA cut-off values specific to the Turkish population using the Sergi formula, which was suggested by the European Working Group on Sarcopenia in Older People (EWGSOP).

Independent risk factors for COVID-19-associated coagulopathy.

OBJECTIVE: Past three years since the beginning of the outbreak, we have obtained satisfactory data on COVID-19. However, data on risk factors of COVID-19-associated coagulopathy (CAC) are extremely limited. Prediction of CAC might be a game changer since it is related to poor prognosis. Seeking independent risk factors for CAC was the main aim of the study. PATIENTS AND METHODS: 510 hospitalized COVID-19 patients were retrospectively screened. Forty-eight of them were excluded due to irrelevant D-dimer or ferritin elevation. The remaining patients were stratified into three groups as overt coagulopathy, significant pulmonary microthrombosis, and patients without coagulopathy. The overt coagulopathy group included cases with macrothrombosis or disseminated intravascular coagulation (DIC). The significant pulmonary microthrombosis group covered the cases that had clinical deterioration with simultaneous marked D-dimer elevation. The group of patients without coagulopathy included the asymptomatic patients with normal or elevated D-dimer levels. RESULTS: Overt coagulopathy developed in 3.2% and significant pulmonary microthrombosis in 10.1% of the patients. In the multivariate analysis, not receiving low molecular weight heparin (LMWH) (p=0.002), a level of D-dimer >15,000 U/ml (p=0.013) were associated with overt coagulopathy. In addition, levels of initial LDH >480 IU/L (p=0.022) and initial ferritin >1,000 ng/ml (p=0.036) were associated with significant pulmonary microthrombosis. Not receiving LMWH (p=0.001) was also associated with significant pulmonary microthrombosis, when multivariate analysis was performed by the parameters with a p-value <0.1 in the univariate analysis. Furthermore, all cases with DIC had Gram-negative bacterial sepsis. CONCLUSIONS: Not receiving LMWH, high levels of D-dimer, initial LDH, and initial ferritin are independent risk factors for CAC. DIC does not appear to develop based on COVID-19.

Malnutrition risk in hospitalized patients measured with Nutrition Risk Screening 2002 tool and its association with in-hospital mortality.

OBJECTIVE: Malnutrition is related to increased morbidity, mortality, and costs. NRS-2002 is a practical malnutrition risk (MR) screening tool approved by the European Society for Clinical Nutrition and Metabolism (ESPEN) for inpatients. We aimed to reveal the inpatient MR using NRS-2002, and to examine the relationship between MR and in-hospital mortality. PATIENTS AND METHODS: The results of inpatient nutritional screening in a tertiary referral center university hospital were retrospectively analyzed. The NRS-2002 test was used for defining MR. Comorbidities, initial and follow-up anthropometric data, NRS-2002 score, food intake, weight status, and laboratory analysis were examined. In-hospital mortality was noted. RESULTS: Data from 5,999 patients were evaluated. On admission, 49.8% of the patients had MR, and 17.3% had severe MR (sMR). MR-sMR was higher in geriatric patients (62.0-28.5%). Those with dementia had the highest MR (71%), followed by stroke (66%) and malignancy (62%). Age and serum C-reactive protein (CRP) were higher, and body weight, BMI, serum albumin, and creatinine were lower in patients with MR. Multivariate analysis showed that age, albumin, CRP, congestive heart failure (CHF), malignancy, dementia, and stroke were independently associated with MR. The overall mortality rate during hospitalization was 7.9%. MR was associated with mortality regardless of serum CRP, albumin, body mass index (BMI), and age. Half of the patients received nutritional treatment (NT). NT resulted in preserved or increased body weight and albumin levels among patients and the geriatric group with MR. CONCLUSIONS: AMR revealed that NRS-2002 is positive in approximately half of the hospitalized patients, which is associated with in-hospital mortality independent of the underlying diseases. NT is related to weight gain and increased serum albumin.

Chemoenzymatic synthesis and utilization of a SAM analog with an isomorphic nucleobase.

SalL, an enzyme that catalyzes the synthesis of SAM from l-methionine and 5'-chloro-5'-deoxyoadenosine, is shown to accept 5'-chloro-5'-deoxythienoadenosine as a substrate and facilitate the synthesis of a synthetic SAM analog with an unnatural nucleobase. This synthetic cofactor is demonstrated to replace SAM in the DNA methylation reaction with M.TaqI.

Activity of the HMGB1-derived immunostimulatory peptide Hp91 resides in the helical C-terminal portion and is enhanced by dimerization.

We have previously shown that an 18 amino acid long peptide, named Hp91, whose sequence corresponds to a region within the endogenous protein HMGB1, activates dendritic cells (DCs) and acts as adjuvant in vivo by potentiating Th1-type antigen-specific immune responses. We analyzed the structure-function relationship of the Hp91 peptide to investigate the amino acids and structure responsible for immune responses. We found that the cysteine at position 16 of Hp91 enabled formation of reversible peptide dimmers, monomer and dimmer were compared for DC binding and activation. Stable monomers and dimers were generated using a maleimide conjugation reaction. The dimer showed enhanced ability to bind to and activate DCs. Furthermore, the C-terminal 9 amino acids of Hp91, named UC1018 were sufficient for DC binding and Circular dichroism showed that UC1018 assumes an alpha-helical structure. The ninemer peptide UC1018 induced more potent antigen-specific CTL responses in vivo as compared to Hp91 and it protected mice from tumor development when used in a prophylactic vaccine setting. We have identified a short alpha helical peptide that acts as potent adjuvant inducing protective immune responses in vivo.

Chemopreventive potential of Annona muricata L leaves on chemically-induced skin papillomagenesis in mice.

Annona muricata L (Annonaceae), commonly known as soursop has a long, rich history in herbal medicine with a lengthy recorded indigenous use. It had also been found to be a promising new anti-tumor agent in numerous in vitro studies. The present investigation concerns chemopreventive effects in a two-stage model of skin papillomagenesis. Chemopreventive effects of an ethanolic extract of A. muricata leaves (AMLE) was evaluated in 6-7 week old ICR mice given a single topical application of 7,12-dimethylbenza(α)anthracene (DMBA 100 µg/100 µl acetone) and promotion by repeated application of croton oil (1% in acetone/ twice a week) for 10 weeks. Morphological tumor incidence, burden and volume were measured, with histological evaluation of skin tissue. Topical application of AMLE at 30, 100 and 300 mg/kg significantly reduced DMBA/croton oil induced mice skin papillomagenesis in (i) peri-initiation protocol (AMLE from 7 days prior to 7 days after DMBA), (ii) promotion protocol (AMLE 30 minutes after croton oil), or (iii) both peri-initiation and promotion protocol (AMLE 7 days prior to 7 day after DMBA and AMLE 30 minutes after croton oil throughout the experimental period), in a dose dependent manner (p<0.05) as compared to carcinogen-treated control. Furthermore, the average latent period was significantly increased in the AMLE-treated group. Interestingly, At 100 and 300 mg/ kg, AMLE completely inhibited the tumor development in all stages. Histopathological study revealed that tumor growth from the AMLE-treated groups showed only slight hyperplasia and absence of keratin pearls and rete ridges. The results, thus suggest that the A.muricata leaves extract was able to suppress tumor initiation as well as tumor promotion even at lower dosage.

The ribosomal A-site as an inspiration for the design of RNA binders.

The specific binding of aminoglycoside antibiotics to the bacterial ribosomal decoding site (A-site) has inspired the study of RNA-small molecules interactions and the search for novel RNA binders. Among the numerous RNA targets studied so far, the A-site holds a unique place. It is among the few truly validated RNA targets for which naturally occurring ligands have been discovered as "cognate" binders. In addition, due to its encapsulating architecture, the A-site is a more discriminating RNA target when compared to other RNA sequences. Previous observations and current challenges for the designers of potent and specific RNA binders are discussed.

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes.

The design, synthesis and study of internally fluorescent hammerhead (HH) ribozymes, where changes in fluorescence parameters directly reflect the progress of the ribozyme's cleavage chemistry, are described. The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster. The incorporation of 2AP, an unnatural nucleoside, does not interfere with the ribozyme folding and catalysis. Since 2AP is highly sensitive to environmental changes, its fluorescence is dramatically altered upon ribozyme-mediated cleavage of the substrate. This generates a measurable signal that directly reflects the progress of the ribozyme's reaction in real time. Identical pseudo first order rate constants are obtained for HH constructs using both continuous fluorescence monitoring and radioactive labeling. This rapid and real-time monitoring facilitates the study of ribozyme activity under different conditions (e.g., ionic strength, pH, etc.), and provides a useful assay to rapidly screen potential inhibitors. Three hitherto unknown HH inhibitors are presented and compared to neomycin B and chlortetracycline, two previously studied HH inhibitors. All three new small molecules, neo-acridine, guanidino-neomycin B, and [Delta-(Eilatin)Ru(bpy)(2)](2+), prove to be better inhibitors than neomycin B or chlortetracycline. Investigating HH inhibition under different ionic strengths reveals that the binding of neo-acridine, [Delta-(Eilatin)Ru(bpy)(2)](2+), and chlortetracycline to the HH involves hydrophobic interactions as their RNA affinities are largely unaffected by increasing salt concentrations. In contrast, neomycin B loses more than 50-fold of its inhibitory ability as the NaCl concentration is increased from 50 to 500mM.

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif.

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.

Synthesis and anti-HIV activity of guanidinoglycosides.

The new guanidinylation reagent N,N'-diBoc-N''-triflylguanidine was used to efficiently convert multiamine-containing glycosides including kanamycin A and B, tobramycin, paromomycin, and neomycin B to the corresponding fully guanidinylated analogues (guanidinoglycosides). This transformation occurs in the presence of H(2)O under mild conditions. Guanidinotobramycin and guanidinoneomycin B were found to inhibit the replication of the HIV virus with activities approximately 100 times greater than the parent aminoglycosides.

Conjugated 1,10-Phenanthrolines as Tunable Fluorophores.

Multiple emission colors can be generated with the same compound from a novel family of highly emissive and visibly fluorescent 1,10-phenanthrolines 1. The emission wavelength of any derivative is dictated by the nature of its substituent and can be further modulated by exogenous additives such as protons or metal ions. R=H, Me, OMe, NMe(2).

tRNA(Phe) binds aminoglycoside antibiotics.

Aminoglycoside antibiotics have recently been found to bind to a variety of unrelated RNA molecules, including sequences that are important for retroviral replication. We report the binding of neomycin B, kanamycin A, and Neo-Neo (a synthetic neomycin-neomycin dimer) to tRNA(Phe). Using thermal denaturation studies, fluorescence spectroscopy, Pb2+-mediated tRNA(Phe) cleavage, and gel mobility shift assays, we have established that aminoglycosides interact with yeast tRNA(Phe) and are likely to induce a conformational change. Thermal denaturation studies revealed that aminoglycosides have a substantial stabilizing effect on tRNA(Phe) secondary and tertiary structures, much greater than the stabilization effect of spermine, an unstructured polyamine. Aminoglycoside-induced inhibition of Pb2+-mediated tRNA(Phe) cleavage yielded IC50 values of: 5 microM for Neo-Neo, 100 microM for neomycin B, > 1 mM for kanamycin A, and > 10 mM for spermine. Enzymatic and chemical footprinting indicate that the anticodon stem as well as the junction of the TpsiC and D loops are preferred aminoglycoside binding sites.

Enhanced RNA binding of dimerized aminoglycosides.

Aminoglycoside antibiotics have recently emerged as an intriguing family of RNA binding molecules and they became leading structures for the design of novel RNA ligands. The demystification of the aminoglycoside-RNA recognition phenomenon is required for the development of superior binders. To explore the existence of multiple binding sites in a large RNA molecule, we have synthesized covalently linked symmetrical and nonsymmetrical dimeric aminoglycosides. These unnatural derivatives were compared to their natural "monomeric" counterparts in their ability to inhibit the Tetrahymena ribozyme. The dimeric aminoglycosides inhibit ribozyme function 20 to 1.2 x 10(3) fold more effectively than their natural parent compounds. The inhibition curves of dimeric aminoglycosides have characteristic shapes suggesting the presence of at least two high affinity-binding sites within the ribozyme's three-dimensional fold. The interaction of a dimeric aminoglycoside with two complementary sites of the RNA molecule is proposed. This binding motif may have implications on the development of new drugs targeting pivotal RNA molecules of bacterial and viral pathogens.

Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme.

Aminoglycoside antibiotics inhibit protein biosynthesis and various ribozymes. Structural electrostatic complementarity can explain the inhibition mechanism of the hammerhead ribozyme: positively charged ammonium groups match the negatively charged metal-ion-binding pockets created by the RNA fold's electrostatic field.

Tobramycin-EDTA conjugate: a noninnocent affinity-cleaving reagent.

The design, synthesis, and ribozyme inhibitory activity of a novel EDTA-aminoglycoside conjugate are reported. This affinity cleaving reagent is a noninnocent RNA binder: its RNA affinity, judged by its ability to inhibit the hammerhead ribozyme HH16, is different than the parent natural product and is markedly dependent on the oxidation state of the chelated metal ion.

Vibrational CD studies of interchain hydrogen-bonded tripodal peptides.

The solution conformations of three trispeptides--L,L,L-1,3,5-C6H3[CH2NHCOCH(X)-NHBoc++ +]3, X = CH3 (Ala) or CH2CH(CH3)2 (Leu), and L,L,L-N(CH2CH2NHCOCH[CH2-CH(CH3)2]NHBoc)3--have been determined from their ir and vibrational CD (VCD) spectra in the NH stretching and carbonyl stretching regions in apolar solution. The compounds containing L-Leu are shown to occur primarily in a propeller conformation with C3 symmetry that is stabilized by interchain hydrogen bonds. Through application of the coupled oscillator model of VCD, a right-handed sense for the hydrogen-bonded chains in the propeller is deduced, in agreement with previous empirical force field calculations. The spectra also provide evidence for interchain association between two chains, resulting in a C10-ring. For chains not involved in interchain association, the spectra reveal the presence of C7-rings within a chain. The trispeptide containing L-Ala is found to occur primarily in a random form.

Assessment of the purity of d,l HM-PAO from diastereomeric mixtures using NMR techniques.

We have shown that 1H NMR can serve as a suitable analytical tool for the assessment of relative amounts of d,l and meso HM-PAO isomers in a diastereomeric mixture and for the determination of the purity of either one of the two diastereomers alone. Therefore, we believe that this method will be useful for quality control in both academic and government regulatory laboratories as well as for radiopharmaceutical manufacturers. In addition, this analytical tool can provide rapid screening of possible suitable stereospecific reducing agents for the preferential formation of the d,l HM-PAO diastereomer without the necessity of completely working up the reduction reaction mixture.

Synthetic ferrichrome analogues with growth promotion activity for Arthrobacter flavescens.

Two families of trihydroxamic acid analogues of ferrichrome were chemically synthesized and tested for biological activity with Arthrobacter flavescens. Compounds using a tertiary amine as anchor showed little activity. Several compounds using tetrahedral carbon as anchor showed activity approaching or equalling that of the natural siderophore, ferrichrome. The biological activity is discussed in relation to physical and chemical properties of the analogues.