No massive black holes in the Milky Way halo.

The gravitational wave detectors have unveiled a population of massive black holes that do not resemble those observed in the Milky Way1-3 and whose origin is debated4-6. According to one possible explanation, these black holes may have formed from density fluctuations in the early Universe (primordial black holes)7-9, and they should comprise from several to 100% of dark matter to explain the observed black hole merger rates10-12. If such black holes existed in the Milky Way dark matter halo, they would cause long-timescale gravitational microlensing events lasting years13. The previous experiments were not sufficiently sensitive to such events14-17. Here we present the results of the search for long-timescale microlensing events among the light curves of nearly 80 million stars located in the Large Magellanic Cloud that were monitored for 20 years by the OGLE survey18. We did not find any events with timescales longer than one year, whereas all shorter events detected may be explained by known stellar populations. We find that compact objects in the mass range from 1.8 × 10-4 to 6.3 M⊙ cannot compose more than 1% of dark matter, and those in the mass range from 1.3 × 10-5 to 860 M⊙ cannot make up more than 10% of dark matter. Thus, primordial black holes in this mass range cannot simultaneously explain a significant fraction of dark matter and gravitational wave events.

Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

Risk factors for bariatric surgery in patients over 65 years of age-a multicenter retrospective cohort study.

PURPOSE: Societies are aging, life expectancy is increasing, and as a result, the percentage of elderly people in the population is constantly increasing. When qualifying patients over 65 years of age for bariatric surgery, the benefits and risks should be carefully assessed. Weighing risk factors against each other to improve the quality of life and better control of obesity-related diseases. The study aimed to determine risk factors for bariatric surgery among patients over 65 years of age. METHODS: A multicenter, retrospective analysis of patients undergoing laparoscopic bariatric procedures from 2008 to 2022. The patients were divided into two groups: complicated (C) and uncomplicated (UC). Uni- and multivariate logistic regression analysis was performed to obtain significant, independent risk factors. RESULTS: There were 20 (7.0%) patients in C group and 264 (93.0%) patients in UC group. The most common complication was intraperitoneal bleeding (8, 2.8). There was no postoperative mortality. The mean follow-up was 47.5 months. In a multivariate logistic regression analysis, length of stay and %EWL significantly corresponded to general complications (OR 1.173, OR 1.020). A higher weight loss before surgery lowered the risk for hemorrhagic events after surgery (OR 0.889). A longer length of stay corresponded to leak after surgery (OR 1.175). CONCLUSIONS: Bariatric and metabolic surgery appears to be a safe method of obesity treatment in patients over 65 years of age. The most common complication was intraperitoneal bleeding. A prolonged hospital stay may increase the risk of leakage, while a higher weight loss before the surgery may lower the risk of bleeding.

In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing.

The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5'-3' exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria.

No large population of unbound or wide-orbit Jupiter-mass planets.

Planet formation theories predict that some planets may be ejected from their parent systems as result of dynamical interactions and other processes. Unbound planets can also be formed through gravitational collapse, in a way similar to that in which stars form. A handful of free-floating planetary-mass objects have been discovered by infrared surveys of young stellar clusters and star-forming regions as well as wide-field surveys, but these studies are incomplete for objects below five Jupiter masses. Gravitational microlensing is the only method capable of exploring the entire population of free-floating planets down to Mars-mass objects, because the microlensing signal does not depend on the brightness of the lensing object. A characteristic timescale of microlensing events depends on the mass of the lens: the less massive the lens, the shorter the microlensing event. A previous analysis of 474 microlensing events found an excess of ten very short events (1-2 days)-more than known stellar populations would suggest-indicating the existence of a large population of unbound or wide-orbit Jupiter-mass planets (reported to be almost twice as common as main-sequence stars). These results, however, do not match predictions of planet-formation theories and surveys of young clusters. Here we analyse a sample of microlensing events six times larger than that of ref. 11 discovered during the years 2010-15. Although our survey has very high sensitivity (detection efficiency) to short-timescale (1-2 days) microlensing events, we found no excess of events with timescales in this range, with a 95 per cent upper limit on the frequency of Jupiter-mass free-floating or wide-orbit planets of 0.25 planets per main-sequence star. We detected a few possible ultrashort-timescale events (with timescales of less than half a day), which may indicate the existence of Earth-mass and super-Earth-mass free-floating planets, as predicted by planet-formation theories.

Human EXOG Possesses Strong AP Hydrolysis Activity: Implication on Mitochondrial DNA Base Excision Repair.

Most oxidative damage on mitochondrial DNA is corrected by the base excision repair (BER) pathway. However, the enzyme that catalyzes the rate-limiting reaction─deoxyribose phosphate (dRP) removal─in the multienzymatic reaction pathway has not been completely determined in mitochondria. Also unclear is how a logical order of enzymatic reactions is ensured. Here, we present structural and enzymatic studies showing that human mitochondrial EXOG (hEXOG) exhibits strong 5'-dRP removal ability. We show that, unlike the canonical dRP lyases that act on a single substrate, hEXOG functions on a variety of abasic sites, including 5'-dRP, its oxidized product deoxyribonolactone (dL), and the stable synthetic analogue tetrahydrofuran (THF). We determined crystal structures of hEXOG complexed with a THF-containing DNA and with a partial gapped DNA to 2.9 and 2.1 Šresolutions, respectively. The structures illustrate that hEXOG uses a controlled 5'-exonuclease activity to cleave the third phosphodiester bond away from the 5'-abasic site. This study provides a structural basis for hEXOG's broad spectrum of substrates. Further, we show that hEXOG can set the order of BER reactions by generating an ideal substrate for the subsequent reaction in BER and inhibit off-pathway reactions.

The awakening of a classical nova from hibernation.

Cataclysmic variable stars-novae, dwarf novae, and nova-likes-are close binary systems consisting of a white dwarf star (the primary) that is accreting matter from a low-mass companion star (the secondary). From time to time such systems undergo large-amplitude brightenings. The most spectacular eruptions, with a ten-thousandfold increase in brightness, occur in classical novae and are caused by a thermonuclear runaway on the surface of the white dwarf. Such eruptions are thought to recur on timescales of ten thousand to a million years. In between, the system's properties depend primarily on the mass-transfer rate: if it is lower than a billionth of a solar mass per year, the accretion becomes unstable and the matter is dumped onto the white dwarf during quasi-periodic dwarf nova outbursts. The hibernation hypothesis predicts that nova eruptions strongly affect the mass-transfer rate in the binary, keeping it high for centuries after the event. Subsequently, the mass-transfer rate should significantly decrease for a thousand to a million years, starting the hibernation phase. After that the nova awakes again-with accretion returning to the pre-eruption level and leading to a new nova explosion. The hibernation model predicts cyclical evolution of cataclysmic variables through phases of high and low mass-transfer. The theory gained some support from the discovery of ancient nova shells around the dwarf novae Z Camelopardalis and AT Cancri, but direct evidence for considerable mass-transfer changes prior, during and after nova eruptions has not hitherto been found. Here we report long-term observations of the classical nova V1213 Cen (Nova Centauri 2009) covering its pre- and post-eruption phases and precisely documenting its evolution. Within the six years before the explosion, the system revealed dwarf nova outbursts indicative of a low mass-transfer rate. The post-nova is two orders of magnitude brighter than the pre-nova at minimum light with no trace of dwarf nova behaviour, implying that the mass-transfer rate increased considerably as a result of the nova explosion.

Phenolic Composition of Inflorescences of Carduus nutans L.

Carduus nutans L. (Compositae) has been extensively used for medicinal purposes. As other representatives of the genus it is implemented in the treatment of liver disorders and used as diuretic and digestive agent. Previous studies have determined the predominant classes of secondary metabolites in Carduus species. Among the major representatives of their extracts flavonoids, phenolic acids, coumarins, sterols and terpenes were identified. The antiradical capacity of three extracts assessed in the DPPH test revealed the highest radical scavenging properties of methanol extract (the EC50 618±10.03 µg/mL) and based on these results it was selected for phenolic content determination. (TPC=61.49 mg/g). To understand better the induced pharmacological effects of the plant the aim of the study was to determine the composition of the methanol extract from the inflorescences of C. nutans and to study their phenolic composition. In order to determine the composition in a more efficient way, the crude extract was fractionated and subjected to hydrolyses. As a result more than twenty phenolic acids and flavonoids were identified in the extract and fractions by HPLC-DAD and/or HPLC-ESI-TOF-MS. The total extract was later subjected to fractionation by centrifugal partition chromatography using the Arizona system composed of hexane/ethyl acetate/methanol/water (0.7 : 4 : 0.8 : 4 v/v/v/v) to produce fractions enriched in flavonoids that are of high pharmacological significance.

Succinate Dehydrogenase-Deficient Renal Cancer Featuring Fructose-1,6-Biphosphatase Loss, Pyruvate Kinase M2 Overexpression, and SWI/SNF Chromatin Remodeling Complex Aberrations: A Rare Case Report.

Succinate dehydrogenase (SDH)-deficient renal cancer is a rare renal cancer subtype recently accepted by the World Health Organization as a unique subtype of renal cell carcinoma (RCC). Here we report a case of 17-year-old man. The detailed evaluation indicated occurrence of the SDHB-deficient RCC. The genetic testing revealed no germline mutation in SDH genes. Immunohistochemistry showed SDHB deficiency, overexpression of pyruvate kinase M2 and dramatic downregulation of fructose-1,6-bisphosphatase metabolic enzymes, and unaltered levels of phosphorylated AMP-activated protein kinase and mammalian target of rapamycin. Strong upregulation of INI1 and BRG1 and overexpression of BAF180, subunits of SWI/SNF ATP-dependent chromatin remodeling complex, were also found. The identified tumor pathologically did not resemble clear cell renal cell carcinoma (ccRCC), but some metabolic alterations are common for both cancer types. Thus, we postulate that the phenotypical differences between ccRCC and SDHB-deficient RCC may be related to distinct molecular and metabolic alterations. IMPLICATIONS FOR PRACTICE: Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is a rare renal tumor occurring even in young patients. Until now, in all described and genetically tested cases, mutations and deletions in SDH genes have been found. This article describes SDHB-deficient RCC without any germline mutations in SDH genes. Therefore, genetic analysis for germline mutations in SDH genes in SDH-deficient RCC, especially in young individuals, should be strongly recommended, although as of now it is not obligatory. This knowledge will allow improvement of patient monitoring including both disease recurrence and new cancer appearance.

SWI/SNF chromatin remodeling complex and glucose metabolism are deregulated in advanced bladder cancer.

Bladder cancer (BC) is a frequently diagnosed malignancy affecting predominantly adult and elderly populations. It is expected that due to the longer life time, BC will become even more frequent in the future; thus in consequence, it will represent serious health problem of older society part. The treatment of advanced BC is mostly ineffective due to its very aggressive behavior. So far, no effective targeted therapy is used for BC treatment. Here, we found that BC is characterized by lower protein levels of BRM, INI1, and BAF155 main subunits of SWI/SNF chromatin remodeling complex (CRC) which is involved in global control of gene expression and influences various important cellular processes like: cell cycle control, apoptosis, DNA repair, etc. Moreover, the expression of SMARCA2, a BRM encoding gene, strongly correlated with BC metastasis and expression of such metabolic genes as PKM2 and PRKAA1. Furthermore, the analysis of T24 and 5637 commonly used BC cell lines revealed different expression levels of metabolic genes including FBP1 gene encoding Frutose-1,6-Bisphosphatase, an enzyme controlling glycolysis flux and gluconeogenesis. The tested BC cell lines exhibited various molecular and metabolic alterations as well as differential glucose uptake, growth rate, and migration potential. We have shown that BRM subunit is involved in the transcriptional control of genes encoding metabolic enzymes. Moreover, we found that the FBP1 expression level and the SWI/SNF CRCs may serve as markers of molecular subtypes of BC. Collectively, this study may provide a new knowledge about the molecular and metabolic BC subtypes which likely will be of high importance for the clinic in the future.

Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase.

The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity.

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ.

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3'-dideoxy-5-fluoro-3'-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3'-dideoxy-3'-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.

The DNA Polymerase Gamma R953C Mutant Is Associated with Antiretroviral Therapy-Induced Mitochondrial Toxicity.

We found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-γ) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-γ mutant binding affinity for dCTP is 8-fold less than that of the wild type. The R953C mutant shows a 4-fold decrease in discrimination of analog nucleotides relative to the wild type. R953 is located on the "O-helix" that forms the substrate deoxynucleoside triphosphate (dNTP) binding site; the interactions of R953 with E1056 and Y986 may stabilize the O-helix and affect polymerase activity.

[Addiction to benzodiazepines - analysis of patients of Independent Public Hospital for Mental Diseases Patients in Miedzyrzecz].

Benzodiazepines (BZD) are substances with proven anxiolytic, sedative, hypnotic, muscle relaxant, and anticonvulsant effect whose activity targets a macromolecular complex comprising GABAA receptors, benzodiazepine receptors and chloride channels. Broad spectrum of action of benzodiazepines affects their more and more frequent consumption by the patients, despite the reports on their addictive potential. The aim of the study was to analyze patients addicted to benzodiazepines, taking into account factors that may increase the risk of addiction. Material and Methods: The study was based on medical records of 52 patients (27 women, 25 men) of Independent Public Hospital for Mental Diseases Patients in Miedzyrzecz from January 2013 to June 2015. The initial diagnosis of admitted patients included psychiatric and behavioral disorders due to taking hypnotics and sedatives (substance withdrawal). We analyzed the amount and time of use of benzodiazepines, alcohol consumption, as well as previous therapies due to alcohol abuse or alcohol dependence. Results: Among the 52 patients (27 women, 25 men aged 26 to 68 years), the majority of city dwellers was working with secondary education Average time of benzodiazepine use was 16 years, 60% of the patients were addicted to 1 benzodiazepine, 20% to two, 10% to three, and nearly 10% to 4. Clonazepam, alprazolam and diazepam were among the most frequently abused benzodiazepines. Conclusions: More and more often observed too long term treatment with benzodiazepines poses the risk of dependence on this group of drugs.

[Efficacy of alcohol withdrawal syndrome therapy in patients from Independent Public Hospital for Mental Diseases in Miedzyrzecz]. / Skutecznosc leczenia alkoholowego zespofu abstynencyjnego u pacjentów Samodzielnego Publicznego Szpitala dla Nerwowo i Psychicznie Chorych w Miedzyrzeczu.

Consumption of alcohol is a serious social problem. Research on alcohol addicts prove that its consumption affects the physical and mental health of drinking person, his/her family and the social dimension (eg. crime, unemployment, poverty). The aim of this study was to evaluate the effectiveness of the treatment of alcohol withdrawal syndrome (AW) in patients of 2417 Unit of Treatment of Alcohol Withdrawal Syndromes of Independent Public Hospital for Mental Diseases (SPSNPCH) in Miedzyrzecz. The study was conducted in 122 of 24/7 Unit of Treatment of Alcohol Withdrawal Syndromes (SPSNPCH) treated from January to March 2015. Patients during hospitalization were subjected to intensive pharmacotherapy of AW (Stage I) and cognitive-behavioral therapy (Stage II). Of the group of 122 people starting treatment Stage I was completed by 112 patients (90%); 10 patients (8%) have been discharged at their own request. The participation in Stage II was consented only by 54 patients, of which 6 (4%) withdrew from this form of therapy. Full two-stage treatment consisting of pharmacotherapy of AWS and then psychotherapy was completed only by 48 (39%) patients.

Human lung vascular endothelium may limit infection with HRV16 via IFN-ß-dependent mechanisms.

Human rhinovirus 16 (HRV16) may induce inflammatory and antiviral responses in the human lung vascular endothelium (ECs) and impair its barrier functions after infection. However, ECs may regain barrier and metabolic functions. Mechanisms of limitation of HRV16 infection in the lung vascular endothelium are unknown. Human lung vascular endothelium (HMVEC-L) was infected with HRV16. IFN-ß, OAS-1, and PKR expression was assessed by real-time PCR, flow cytometry, and confocal microscope. To prove the significance of IFN-ß in the limitation of HRV16 replication, HMVEC-Ls were preincubated with anti-IFN-ß Abs. To prove the involvement of OAS-1 and PKR in the IFN-dependent limitation of HRV16 replication, HMVEC-Ls were transfected with respective siRNA. HRV16 stimulated IFN-ß production and activated intracellular mechanisms of antiviral immunity based on OAS-1 and PKR activation. Blocking of IFN-ß contributed to the inhibition of intracellular mechanisms of antiviral immunity (OAS-1, PKR) and boosted replication of HRV16. Effective OAS-1 silencing by siRNA caused the increase of HRV16 copy numbers after HRV16 infection. siRNA upregulated the other genes related to the antiviral response. The infected lung vascular endothelium may limit the HRV16 infection. This limitation may be associated with the induction of IFN-ß-dependent intracellular mechanisms based on OAS-1 and PKR activity.

Effect of Primary Versus Revisional One Anastomosis Gastric Bypass (OAGB) on Fatty Acid Profile.

INTRODUCTION: One anastomosis gastric bypass (OAGB) is one option of a revisional procedure for failed sleeve gastrectomy. Moreover, it can be used as a primary bariatric procedure, and is an effective surgery resulting in significant weight loss and the resolution or improvement of obesity-associated medical problems, accompanied by low perioperative complications. However, as with any therapy, OAGB has its limitations, including micronutrient deficiency or malnutrition. In our study, we compared the fatty acid (FA) profile in serum of patients after both primary OAGB (pOAGB) and revisional OAGB (rOAGB) to identify potential postsurgical FA alterations. METHODS: This is a retrospective study on patients with obesity who underwent OAGB procedures (pOAGB n=68; rOAGB n=17), conducted from 2016 to 2018. In blood, we analyzed a series of biochemical parameters, and in the serum, the FA profile was determined using gas chromatography-mass spectrometry. RESULTS: The percentage of excess BMI loss (% EBMIL) after pOAGB was 73.5 ± 2.47% in comparison to 45.9 ± 4.15% in the rOAGB group (p<0.001). In contrast to the lack of effect of rOAGB on most polyunsaturated FAs, in the pOAGB group, there was a decrease in eicosapentaenoic acid, and eicosatetraenoic and docosahexaenoic acid levels (p<0.001). We also found a decrease in very long-chain FAs (VLCFAs) and an increase in branched-chain FAs (BCFAs) after both types of OAGB procedure. CONCLUSIONS: Both OAGB procedures improved the profile of most FAs, leading to a decrease in VLCFAs, which are considered harmful, and an improvement in BCFAs, which are considered to be beneficial. There is a need to further investigate the possibility of n-3 polyunsaturated FA supplementation after pOAGB, due to the large decrease in these FAs after pOAGB.

The outcomes of Re-Redo bariatric surgery-results from multicenter Polish Revision Obesity Surgery Study (PROSS).

The increasing prevalence of bariatric surgery has resulted in a rise in the number of redo procedures as well. While redo bariatric surgery has demonstrated its effectiveness, there is still a subset of patients who may not derive any benefits from it. This poses a significant challenge for bariatric surgeons, especially when there is a lack of clear guidelines. The primary objective of this study is to evaluate the outcomes of patients who underwent Re-Redo bariatric surgery. We conducted a retrospective cohort study on a group of 799 patients who underwent redo bariatric surgery between 2010 and 2020. Among these patients, 20 individuals underwent a second elective redo bariatric surgery (Re-Redo) because of weight regain (15 patients) or insufficient weight loss, i.e. < 50% EWL (5 patients). Mean BMI before Re-Redo surgery was 38.8 ± 4.9 kg/m2. Mean age was 44.4 ± 11.5 years old. The mean %TWL before and after Re-Redo was 17.4 ± 12.4% and %EBMIL was 51.6 ± 35.9%. 13/20 patients (65%) achieved > 50% EWL. The mean final %TWL was 34.2 ± 11.1% and final %EBMIL was 72.1 ± 20.8%. The mean BMI after treatment was 31.9 ± 5.3 kg/m2. Complications occurred in 3 of 20 patients (15%), with no reported mortality or need for another surgical intervention. The mean follow-up after Re-Redo was 35.3 months. Although Re-Redo bariatric surgery is an effective treatment for obesity, it carries a significant risk of complications.

The Escherichia coli primosomal DnaT protein exists in solution as a monomer-trimer equilibrium system.

The oligomerization reaction of the Escherichia coli DnaT protein has been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. In solution, DnaT exists as a monomer-trimer equilibrium system. At the estimated concentration in the E. coli cell, DnaT forms a mixture of the monomer and trimer states with a 3:1 molar ratio. In spite of the modest affinity, the trimerization is a highly cooperative process, without the detectable presence of the intervening dimer. The DnaT monomer consists of a large N-terminal core domain and a small C-terminal region. The removal of the C-terminal region dramatically affects the oligomerization process. The isolated N-terminal domain forms a dimer instead of the trimer. These results indicate that the DnaT monomer possesses two structurally different, interacting sites. One site is located on the N-terminal domain, and two monomers, in the trimer, are associated through their binding sites located on that domain. The C-terminal region forms the other interacting site. The third monomer is engaged through the C-terminal regions. Surprisingly, the high affinity of the N-terminal domain dimer indicates that the DnaT monomer undergoes a conformational transition upon oligomerization, involving the C-terminal region. These data and the high specificity of the trimerization reaction, i.e., lack of any oligomers higher than a trimer, indicate that each monomer in the trimer is in contact with the two remaining monomers. A model of the global structure of the DnaT trimer based on the thermodynamic and hydrodynamic data is discussed.

Energetics of the Escherichia coli DnaT protein trimerization reaction.

Thermodynamic and structural characteristics of the Escherichia coli DnaT protein trimerization reaction have been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. Binding of magnesium to the DnaT monomers regulates the intrinsic affinity of the DnaT trimerization reaction. Comparison between the DnaT trimer and the isolated N-terminal core domain suggests that magnesium binds to the N-terminal domain but does not associate with the C-terminal region of the protein. The magnesium binding process is complex and involves approximately three Mg(2+) cations per protein monomer. The observed effect seems to be specific for Mg(2+). In the examined salt concentration range, monovalent cations and anions do not affect the trimer assembly process. However, magnesium affects neither the cooperativity of the trimerization reaction nor the GnHCl-induced trimer dissociation, strongly indicating that Mg(2+) indirectly stabilizes the trimer through the induced changes in the monomer structures. Nevertheless, formation of the trimer also involves specific conformational changes of the monomers, which are independent of the presence of magnesium. Binding of Mg(2+) cations dramatically changes the thermodynamic functions of the DnaT trimerization, transforming the reaction from a temperature-dependent to temperature-independent process. Highly cooperative dissociation of the trimer by GnHCl indicates that both interacting sites of the monomer, located on the N-terminal core domain and formed by the small C-terminal region, are intimately integrated with the entire protein structure. In the intact protein, the C-terminal region most probably interacts with the corresponding binding site on the N-terminal domain of the monomer. Functional implications of these findings are discussed.