The genetic basis for adaptation of model-designed syntrophic co-cultures.

Understanding the fundamental characteristics of microbial communities could have far reaching implications for human health and applied biotechnology. Despite this, much is still unknown regarding the genetic basis and evolutionary strategies underlying the formation of viable synthetic communities. By pairing auxotrophic mutants in co-culture, it has been demonstrated that viable nascent E. coli communities can be established where the mutant strains are metabolically coupled. A novel algorithm, OptAux, was constructed to design 61 unique multi-knockout E. coli auxotrophic strains that require significant metabolite uptake to grow. These predicted knockouts included a diverse set of novel non-specific auxotrophs that result from inhibition of major biosynthetic subsystems. Three OptAux predicted non-specific auxotrophic strains-with diverse metabolic deficiencies-were co-cultured with an L-histidine auxotroph and optimized via adaptive laboratory evolution (ALE). Time-course sequencing revealed the genetic changes employed by each strain to achieve higher community growth rates and provided insight into mechanisms for adapting to the syntrophic niche. A community model of metabolism and gene expression was utilized to predict the relative community composition and fundamental characteristics of the evolved communities. This work presents new insight into the genetic strategies underlying viable nascent community formation and a cutting-edge computational method to elucidate metabolic changes that empower the creation of cooperative communities

A selective strategy for targeting primary hyperoxaluria diseases

Funding Information: Authors wish to thank the Centro de Instrumentación Científico-Técnica (CICT) of the University of Jaén, Spain, for partial financial support. A.A.-A. is grateful for the postdoctoral fellowship from Fundación Alfonso Martín Escudero. Authors acknowledge the use of the National Facility ELECMI ICTS, node “Laboratorio de Microscopias Avanzadas” at Universidad de Zaragoza. This research has also partially been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (grant RTI2018-098560-B-C22) and by the Andalusian Consejería de Economía y Conocimiento (FEDER program 2014-2020: grant number 1380682). This work was partially supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020). Funding Information: This research has also partially been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (grant RTI2018-098560-B-C22) and by the Andalusian Consejería de Economía y Conocimiento (FEDER program 2014-2020: grant number 1380682). This work was partially supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020). Funding Information: Authors wish to thank the Centro de Instrumentación Científico-Técnica (CICT) of the University of Jaén, Spain, for partial financial support. A.A.-A. is grateful for the postdoctoral fellowship from Fundación Alfonso Martín Escudero. Authors acknowledge the use of the National Facility ELECMI ICTS, node “Laboratorio de Microscopias Avanzadas” at Universidad de Zaragoza. Publisher Copyright: © 2022 The Author(s)Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism that result in an excess of oxalate production by the oxidation of glyoxylate by the human lactate dehydrogenase A enzyme (hLDHA). The selective liver inhibition of this enzyme is one of the therapeutic strategies followed in the treatment of this disease. Even though several efforts have been recently performed using gene silencing by the RNA interference approach, small-molecule inhibitors that selectively reach hepatocytes are preferred since they present the advantages of a lower production cost and better pharmacological properties. In that sense, the design, synthesis, and physicochemical characterization by NMR, FTIR, DLS and TEM of two nanocarriers based on chitosan conjugates (1, non-redox-sensitive; 2, redox-sensitive) have been performed to (i) achieve the selective transport of hLDHA inhibitors into hepatocytes and (ii) their disruption once they reach the hepatocytes cytosol. Polymer 2 self-assembled into micelles in water and showed high drug loadings (19.8–24.5 %) and encapsulation efficiencies (31.9–40.8%) for the hLDHA inhibitors (I-III) tested. The non-redox-sensitive micelle 1 remained stable under different glutathione (GSH) concentrations (10 μM and 10 mM), and just a residual release of the inhibitor encapsulated was observed (less than 10 %). On the other hand, micelle 2 was sufficiently stable under in vitro physiological conditions (10 μM, GSH) but it quickly disassembled under the simulated reducing conditions present inside hepatocytes (10 mM GSH), achieving a 60 % release of the hLDHA inhibitor encapsulated after 24 h, confirming the responsiveness of the developed carrier to the high levels of intracellular GSH.publishersversionpublishe

Two-pore channel 2 (TPC2) modulates store-operated Ca2+ entry

AbstractTwo-pore channels (TPCs) are NAADP-sensitive receptor channels that conduct Ca2+ efflux from the intracellular stores. Discharge of the internal Ca2+ pools results in the activation of store-operated Ca2+ entry (SOCE); however, the role of TPCs in the modulation of SOCE remains unexplored. Mammalian cells express three TPCs: TPC1, TPC2 and TPC3, a pseudogene in humans. Here we report that MEG01 and HEK293 cells endogenously express TPC1 and TPC2. Silencing TPC2 expression results in attenuation of the rate and extent of thapsigargin (TG)-evoked SOCE both in MEG01 and HEK293 cells, without having any effect on the ability of cells to accumulate Ca2+ into the TG-sensitive stores. Similarly, silencing of native TPC2 expression reduced thrombin-induced Ca2+ entry in MEG01 cells. In contrast, silencing of TPC1 expression was without effect either on TG or thrombin-stimulated Ca2+ entry both in MEG01 and HEK293 cells. Biotinylation analysis revealed that TPC1 and TPC2 are expressed in internal membranes. Finally, co-immunoprecipitation experiments indicated that endogenously expressed TPC2, but not TPC1, associates with STIM1 and Orai1, but not with TRPC1, in MEG01 cells with depleted intracellular Ca2+ stores, but not in resting cells. These results provide strong evidence for the modulation of SOCE by TPC2 involving de novo association between TPC2 and STIM1, as well as Orai1, in human cells

Time-Varying Emulator for Short and Long-Term Analysis of Coastal Flood Hazard Potential

Rising seas coupled with ever increasing coastal populations present the potential for significant social and economic loss in the 21st century. Relatively short records of the full multidimensional space contributing to total water level coastal flooding events (astronomic tides, sea level anomalies, storm surges, wave run‐up, etc.) result in historical observations of only a small fraction of the possible range of conditions that could produce severe flooding. The Time‐varying Emulator for Short‐ and Long‐Term analysis of coastal flood hazard potential is presented here as a methodology capable of producing new iterations of the sea‐state parameters associated with the present‐day Pacific Ocean climate to simulate many synthetic extreme compound events. The emulator utilizes weather typing of fundamental climate drivers (sea surface temperatures, sea level pressures, etc.) to reduce complexity and produces new daily synoptic weather chronologies with an auto‐regressive logistic model accounting for conditional dependencies on the El Niño Southern Oscillation, the Madden‐Julian Oscillation, seasonality, and the prior two days of weather progression. Joint probabilities of sea‐state parameters unique to simulated weather patterns are used to create new time series of the hypothetical components contributing to synthetic total water levels (swells from multiple directions coupled with water levels due to wind setup, temperature anomalies, and tides). The Time‐varying Emulator for Short‐ and Long‐Term analysis of coastal flood hazard potential reveals the importance of considering the multivariate nature of extreme coastal flooding, while progressing the ability to incorporate large‐scale climate variability into site specific studies assessing hazards within the context of predicted climate change in the 21st century

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73a oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73a, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention

Gaya Kepemimpinan Perempuan dalam Jabatan Publik (Studi Kasus: Lurah Perempuan di Kelurahan Kesiman Kecamatan Depasar Timur)

Penelitian ini bertujuan untuk mengetahui secara aktual bagaimana gaya kepemimpinan lurah perempuan di Kelurahan Kesiman. Penelitian ini melihat gaya kepemimpinan dari dua sisi yang berbeda yaitu gaya kepemimpinan secara umum dan gaya kepemimpinan khas perempuan. gaya kepemimpinan secara umum terdiri dari gaya kepemimpinan otoriter, demokratis, dan laissez-faire. Sedangkan gaya kepemimpinan khas perempuan terdiri dari maskulin - feminim dan transformasional - transaksional. Metode yang digunakan dalam penelitian ini adalah jenis penelitian kualitatif deskritif. Adapun hasil penelitian menunjukkan bahwa gaya kepemimpinan yang digunakan oleh lurah perempuan di Kesiman adalah gaya kepemimpinan demokratis jika dilihat dari sisi gaya kepemimpinan secara umum, dan gaya kepemimpinan maskulin-transformasional jika dilihat dari gaya kepemimpinan khas perempuan

Robustness and stability in Constraint Programming under dynamism and uncertainty

Many real life problems that can be solved by constraint programming, come from uncertain and dynamic environments. Because of the dynamism, the original problem may change over time, and thus the solution found for the original problem may become invalid. For this reason, dealing with such problems has become an important issue in the fields of constraint programming. In some cases, there exist extant knowledge about the uncertain and dynamic environment. In other cases, this information is fragmentary or unknown. In this paper, we extend the concept of robustness and stability for Constraint Satisfaction Problems (CSPs) with ordered domains, where only limited assumptions need to be made as to possible changes. We present a search algorithm that searches for both robust and stable solutions for CSPs of this nature. It is well-known that meeting both criteria simultaneously is a desirable objective for constraint solving in uncertain and dynamic environments. We also present compelling evidence that our search algorithm outperforms other general-purpose algorithms for dynamic CSPs using random instances and benchmarks derived from real life problems

Neoadjuvant intralesional methotrexate for juvenile xanthogranuloma in an adult

Juvenile xanthogranuloma (JXG) is a non-Langerhans cell histiocytosis usually occurring in infants and typically located in the head or neck.1 Clinically, solitary skin lesions are found in 60%–82% of patients and the most common variant is characterized by one yellowish nodule. Adult onset is rare, and although JXG is usually self-limiting in children, spontaneous resolution is uncommon at older ages. In addition, up to 50% of patients with spontaneous regression develop an atrophy or anetodermal area.2 Thus, complete excision is frequently performed in this population subgroup to achieve better cosmetic results. In disseminated forms, different chemotherapy regimens, corticosteroids and other systemic therapies are used. Herein, we report a case of adult JXG treated with intralesional methotrexate (MTX) resulting in a rapid reduction in size

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance

Finding robust solutions for constraint satisfaction problems with discrete and ordered domains by coverings

Constraint programming is a paradigm wherein relations between variables are stated in the form of constraints. Many real life problems come from uncertain and dynamic environments, where the initial constraints and domains may change during its execution. Thus, the solution found for the problem may become invalid. The search forrobustsolutions for constraint satisfaction problems (CSPs) has become an important issue in the ¿eld of constraint programming. In some cases, there exists knowledge about the uncertain and dynamic environment. In other cases, this information is unknown or hard to obtain. In this paper, we consider CSPs with discrete and ordered domains where changes only involve restrictions or expansions of domains or constraints. To this end, we model CSPs as weighted CSPs (WCSPs) by assigning weights to each valid tuple of the problem constraints and domains. The weight of each valid tuple is based on its distance from the borders of the space of valid tuples in the corresponding constraint/domain. This distance is estimated by a new concept introduced in this paper: coverings. Thus, the best solution for the modeled WCSP can be considered as a most robust solution for the original CSP according to these assumptionsThis work has been partially supported by the research projects TIN2010-20976-C02-01 (Min. de Ciencia e Innovacion, Spain) and P19/08 (Min. de Fomento, Spain-FEDER), and the fellowship program FPU.Climent Aunés, LI.; Wallace, RJ.; Salido Gregorio, MA.; Barber Sanchís, F. (2013). Finding robust solutions for constraint satisfaction problems with discrete and ordered domains by coverings. Artificial Intelligence Review. 1-26. https://doi.org/10.1007/s10462-013-9420-0S126Climent L, Salido M, Barber F (2011) Reformulating dynamic linear constraint satisfaction problems as weighted csps for searching robust solutions. 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