Antitumor activity of the PI3K δ-sparing inhibitor MEN1611 in PIK3CA mutated, trastuzumab-resistant HER2 + breast cancer.

PURPOSE: Dysregulation of the PI3K pathway is one of the most common events in breast cancer. Here we investigate the activity of the PI3K inhibitor MEN1611 at both molecular and phenotypic levels by dissecting and comparing its profile and efficacy in HER2 + breast cancer models with other PI3K inhibitors. METHODS: Models with different genetic backgrounds were used to investigate the pharmacological profile of MEN1611 against other PI3K inhibitors. In vitro studies evaluated cell viability, PI3K signaling, and cell death upon treatment with MEN1611. In vivo efficacy of the compound was investigated in cell line- and patient-derived xenografts models. RESULTS: Consistent with its biochemical selectivity, MEN1611 demonstrated lower cytotoxic activity in a p110δ-driven cellular model when compared to taselisib, and higher cytotoxic activity in the p110ß-driven cellular model when compared to alpelisib. Moreover, MEN1611 selectively decreased the p110α protein levels in PIK3CA mutated breast cancer cells in a concentration- and proteasome-dependent manner. In vivo, MEN1611 monotherapy showed significant and durable antitumor activity in several trastuzumab-resistant PIK3CA-mutant HER2 + PDX models. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. CONCLUSIONS: The profile of MEN1611 and its antitumoral activity suggest an improved profile as compared to pan-inhibitors, which are limited by a less than ideal safety profile, and isoform selective molecules, which may potentially promote development of resistance mechanisms. The compelling antitumor activity in combination with trastuzumab in HER2 + trastuzumab-resistant, PIK3CA mutated breast cancer models is at the basis of the ongoing B-Precise clinical trial (NCT03767335).

Mangrovivirga cuniculi gen. nov., sp. nov., a moderately halophilic bacterium isolated from bioturbated Red Sea mangrove sediment, and proposal of the novel family Mangrovivirgaceae fam. nov.

A strictly aerobic, Gram-stain-negative, non-motile, rod-shaped bacterium, designated strain R1DC9T, was isolated from sediments of a mangrove stand on the Red Sea coast of Saudi Arabia via diffusion chamber cultivation. Strain R1DC9T grew at 20-40 °C (optimum, 37 °C), pH 6-10 (optimum, pH 8) and 3-11 % NaCl (optimum, 7-9 %) in the cultivation medium. The genome of R1DC9T was 4 661 901 bp long and featured a G+C content of 63.1 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence and whole-genome multilocus sequence analysis using 120 concatenated single-copy genes revealed that R1DC9T represents a distinct lineage in the order Cytophagales and the phylum Bacteroidetes separated from the Roseivirgaceae and Marivirgaceae families. R1DC9T displayed 90 and 89 % 16S rRNA gene sequence identities with Marivirga sericea DSM 4125T and Roseivirga ehrenbergii KMM 6017T, respectively. The predominant quinone was MK7. The polar lipids were phosphatidylethanolamine, two unknown phospholipids and two unknown lipids. The predominant cellular fatty acids were the saturated branch chain fatty acids iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 0, along with a low percentage of the monounsaturated fatty acid C16 : 1 ω5c. Based on differences in phenotypic, physiological and biochemical characteristics from known relatives, and the results of phylogenetic analyses, R1DC9T (=KCTC 72349T=JCM 33609T=NCCB 100698T) is proposed to represent a novel species in a new genus, and the name Mangrovivirga cuniculi gen. nov., sp. nov. is proposed. The distinct phylogenetic lineage among the families in the order Cytophagales indicates that R1DC9T represents a new family for which the name Mangrovivirgaceae fam. nov. is proposed.

Kaustia mangrovi gen. nov., sp. nov. isolated from Red Sea mangrove sediments belongs to the recently proposed Parvibaculaceae family within the order Rhizobiales.

We isolated a novel strain, R1DC25T, described as Kaustia mangrovi gen. nov. sp. nov. from the sediments of a mangrove forest on the coast of the Red Sea in Saudi Arabia. This isolate is a moderately halophilic, aerobic/facultatively anaerobic Gram-stain-negative bacterium showing optimum growth at between 30 and 40 °C, at a pH of 8.5 and with 3-5 % NaCl. The genome of R1DC25T comprises a circular chromosome that is 4 630 536 bp in length, with a DNA G+C content of 67.3 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence and whole-genome multilocus sequence analysis of 120 concatenated single-copy genes revealed that R1DC25T represents a distinct lineage within the family Parvibaculaceae in the order Rhizobiales within the class Alphaproteobacteria. R1DC25T showing 95.8, 95.3 and 94.5 % 16S rRNA gene sequence identity with Rhodoligotrophos appendicifer, Rhodoligotrophos jinshengii and Rhodoligotrophos defluvii, respectively. The predominant quinone was Q-10, and the polar lipids were phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, as well as several distinct aminolipids and lipids. The predominant cellular fatty acids were C19 : 0 cyclo ω8c, a combination of C18 : 1ω7c and/or C18 : 1ω6c and C16 : 0. On the basis of the differences in the phenotypic, physiological and biochemical characteristics from its known relatives and the results of our phylogenetic analyses, R1DC25T (=KCTC 72348T;=JCM 33619T;=NCCB 100699T) is proposed to represent a novel species in a novel genus, and we propose the name Kaustia mangrovi gen. nov., sp. nov. (Kaustia, subjective name derived from the abbreviation KAUST for King Abdullah University of Science and Technology; mangrovi, of a mangrove).

Targeting CD205 with the antibody drug conjugate MEN1309/OBT076 is an active new therapeutic strategy in lymphoma models.

Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematological cancers. Here, we report preclinical data on the anti-tumor activity of the first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination and validation experiments on in vivo models. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25/42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax, and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.

Extracellular matrix proteins as diagnostic markers of breast carcinoma.

Changes in amount and composition of extracellular matrix (ECM) are considered a hallmark of tumor development. We tested the hypothesis that abnormal production of ECM components leads to blood-released ECM molecules representing tumor circulating biomarkers. Candidate genes were selected through class comparison in two publicly available datasets and confirmed in paired normal and tumor associated fibroblasts from breast carcinoma (BC) specimens. Production and release of ECM molecules were evaluated in normal human dermal fibroblasts (NHDFs) treated with conditioned media from three BC cell lines. Plasma samples from healthy donors and from patients with malignant or benign breast disease were tested by ELISA for the presence of collagen 11a1 (COL11A1), collagen oligomeric matrix protein (COMP), and collagen 10a1 (COL10A1). Selected ECM molecules were investigated by IHC in malignant and benign specimens. In silico analysis of gene expression profiles identified 11 ECM genes significantly up-regulated in tumor versus normal tissue. Western blot analyses revealed increased levels of molecules encoded by three of these genes, COL11A1, COMP, and COL10A1, in cell lysates and supernatants of conditioned NHDFs. Class comparison and class prediction analyses of two independent series of human plasma samples identified the combination of COL11A1, COMP, and COL10A1 as potentially informative in discriminating BC patients from those with benign disease. The three molecules resulted expressed in the stroma of BC tissue samples. Our results indicate that circulating COL11A1, COMP, and COL10A1 may be useful in diagnostic assessment of suspicious breast nodules and ECM molecules could represent an avenue to biomarker identification.

Tumor-extracellular matrix interactions: Identification of tools associated with breast cancer progression.

Several evidences support the concept that cancer development and progression are not entirely cancer cell-autonomous processes, but may be influenced, and possibly driven, by cross-talk between cancer cells and the surrounding microenvironment in which, besides immune cells, stromal cells and extracellular matrix (ECM) play a major role in regulating distinct biologic processes. Stroma and ECM-related signatures proved to influence breast cancer progression, and to contribute to the identification of tumor phenotypes resistant to cytotoxic and hormonal treatments. The possible clinical implications of the interplay between tumor cells and the microenvironment, with special reference to ECM remodelling, will be discussed in this review.

Complexity in the tumour microenvironment: Cancer associated fibroblast gene expression patterns identify both common and unique features of tumour-stroma crosstalk across cancer types.

Cancer is a complex disease, driven by the accumulation of several somatic aberrations but fostered by a two-way interaction between tumour cells and the surrounding microenvironment. Cancer associated fibroblasts (CAFs) represent one of the major players in tumour-stroma crosstalk. Recent in vitro and in vivo studies, often conducted by employing high throughput approaches, have started unravelling the key pathways involved in their functional effects. This review focus on open challenges in the study of CAF properties and function, highlighting at the same time the existence of common mechanisms as well as peculiarities in different cancer types (breast, prostate and lung cancer). Although still limited by current experimental models, which are unable to deal with the full level of complexity of the tumour microenvironment, a better understanding of these mechanisms may enable the identification of new biomarkers and therapeutic targets, to improve current strategies for cancer diagnosis and treatment.

Model-based prediction of effective target exposure for MEN1611 in combination with trastuzumab in HER2-positive advanced or metastatic breast cancer patients.

MEN1611 is a novel orally bioavailable PI3K inhibitor currently in clinical development for patients with HER2-positive (HER2+) PI3KCA mutated advanced/metastatic breast cancer (BC) in combination with trastuzumab (TZB). In this work, a translational model-based approach to determine the minimum target exposure of MEN1611 in combination with TZB was applied. First, pharmacokinetic (PK) models for MEN1611 and TZB in mice were developed. Then, in vivo tumor growth inhibition (TGI) data from seven combination studies in mice xenograft models representative of the human HER2+ BC non-responsive to TZB (alterations of the PI3K/AkT/mTOR pathway) were analyzed using a PK-pharmacodynamic (PD) TGI model for co-administration of MEN1611 and TZB. The established PK-PD relationship was used to quantify the minimum effective MEN1611 concentration, as a function of TZB concentration, needed for tumor eradication in xenograft mice. Finally, a range of minimum effective exposures for MEN1611 were extrapolated to patients with BC, considering the typical steady-state TZB plasma levels in patients with BC following three alternative regimens (i.v. 4 mg/kg loading dose +2 mg/kg q1w, i.v. 8 mg/kg loading dose +6 mg/kg q3w or s.c. 600 mg q3w). A threshold of about 2000 ng·h/ml for MEN1611 exposure associated with a high likelihood of effective antitumor activity in a large majority of patients was identified for the 3-weekly and the weekly i.v. schedule for TZB. A slightly lower exposure (i.e., 25% lower) was found for the 3-weekly s.c. schedule. This important outcome confirmed the adequacy of the therapeutic dose administered in the ongoing phase 1b B-PRECISE-01 study in patients with HER2+ PI3KCA mutated advanced/metastatic BC.

The identification of the new species Nitratireductor thuwali sp. nov. reveals the untapped diversity of hydrocarbon-degrading culturable bacteria from the arid mangrove sediments of the Red Sea.

Introduction: The geological isolation, lack of freshwater inputs and specific internal water circulations make the Red Sea one of the most extreme-and unique-oceans on the planet. Its high temperature, salinity and oligotrophy, along with the consistent input of hydrocarbons due to its geology (e.g., deep-sea vents) and high oil tankers traffic, create the conditions that can drive and influence the assembly of unique marine (micro)biomes that evolved to cope with these multiple stressors. We hypothesize that mangrove sediments, as a model-specific marine environment of the Red Sea, act as microbial hotspots/reservoirs of such diversity not yet explored and described. Methods: To test our hypothesis, we combined oligotrophic media to mimic the Red Sea conditions and hydrocarbons as C-source (i.e., crude oil) with long incubation time to allow the cultivation of slow-growing environmentally (rare or uncommon) relevant bacteria. Results and discussion: This approach reveals the vast diversity of taxonomically novel microbial hydrocarbon degraders within a collection of a few hundred isolates. Among these isolates, we characterized a novel species, Nitratireductor thuwali sp. nov., namely, Nit1536T. It is an aerobic, heterotrophic, Gram-stain-negative bacterium with optimum growth at 37°C, 8 pH and 4% NaCl, whose genome and physiological analysis confirmed the adaptation to extreme and oligotrophic conditions of the Red Sea mangrove sediments. For instance, Nit1536T metabolizes different carbon substrates, including straight-chain alkanes and organic acids, and synthesizes compatible solutes to survive in salty mangrove sediments. Our results showed that the Red Sea represent a source of yet unknown novel hydrocarbon degraders adapted to extreme marine conditions, and their discovery and characterization deserve further effort to unlock their biotechnological potential.

The activity of a PI3K δ-sparing inhibitor, MEN1611, in non-small cell lung cancer cells with constitutive activation of the PI3K/AKT/mTOR pathway.

Background: Lung cancer remains the leading cause of cancer-related death worldwide. Targeted therapies with tyrosine kinase inhibitors (TKIs) result in improvement in survival for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR). Unfortunately, most patients who initially respond to EGFR-TKI ultimately develop resistance to therapy, resulting in cancer progression and relapse. Combination therapy is today a common strategy for the treatment of tumors to increase the success rate, improve the outcome and survival of patients, and avoid the selection of resistant cancer cells through the activation of compensatory pathways. In NSCLC, the phosphoinositide-3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway has been heavily implicated in both tumorigenesis and the progression of disease. Objectives: In this study, we investigated the efficacy of a PI3K δ-sparing inhibitor, MEN1611, in models of NSCLC sensitive and resistant to EGFR inhibitors (erlotinib and gefitinib) with a wild-type PIK3CA gene. Methods: We performed functional, biochemical, and immunohistochemistry studies. Results: We demonstrated good efficacy of MEN1611 in NSCLC devoid of PIK3CA gene mutations but with constitutive activation of the PI3K/AKT pathway and its synergistic effect with gefitinib both in vitro and in vivo. Conclusions: Overall, this preclinical study indicates that the inhibitor could be a candidate for the treatment of NSCLC with an erlotinib/gefitinib-resistant phenotype and constitutive activation of the PI3K/AKT pathway, a phenotype mimicked by our model system.

Two-stage vs single-stage thermophilic anaerobic digestion: comparison of energy production and biodegradation efficiencies.

Two-stage anaerobic digestion (AD) for integrated biohydrogen and biomethane production from organic materials has been reported to promise higher process efficiency and energy recoveries as compared to traditional one-stage AD. This work presents a comparison between two-stage (reactors R1 and R2) and one-stage (reactor R3) AD systems, fed with identical organic substrates and loading rates, focusing the attention on chemical and microbiological aspects. Contrary to previous experiences, no significant differences in overall energy recovery were found for the two-stage and one-stage AD systems. However, an accumulation in R2 of undegraded intermediate metabolites (volatile fatty acids, ketones, amines, amino acids, and phenols) was observed by GC-MS. These compounds were thought to be both cause and effect of this partial inefficiency of the two-stage system, as confirmed also by the less diverse, and thereby less efficient, population of fermentative bacteria observed (by PCR-DGGE) in R2. The extreme environment of R1 (low pH and high metabolites concentrations) probably acted as selector of metabolic pathways, favoring H(2)-producing bacteria able to degrade such a wide variability of intermediate metabolites while limiting other strains. Therefore, if two-stage AD may potentially lead to higher energy recoveries, further efforts should be directed to ensure process efficiency and stability.

Insights Into the Cultivable Bacterial Fraction of Sediments From the Red Sea Mangroves and Physiological, Chemotaxonomic, and Genomic Characterization of Mangrovibacillus cuniculi gen. nov., sp. nov., a Novel Member of the Bacillaceae Family.

Mangrove forests are dynamic and productive ecosystems rich in microbial diversity; it has been estimated that microbial cells in the mangrove sediments constitute up to 91% of the total living biomass of these ecosystems. Despite in this ecosystem many of the ecological functions and services are supported and/or carried out by microorganisms (e.g., nutrient cycling and eukaryotic-host adaptation), their diversity and function are overlooked and poorly explored, especially for the oligotrophic mangrove of the Red Sea coast. Here, we investigated the cultivable fraction of bacteria associated with the sediments of Saudi Arabian Red Sea mangrove forest by applying the diffusion-chamber-based approach in combination with oligotrophic medium and long incubation time to allow the growth of bacteria in their natural environment. Cultivation resulted in the isolation of numerous representatives of Isoptericola (n = 51) and Marinobacter (n = 38), along with several less abundant and poorly study taxa (n = 25) distributed across ten genera. Within the latest group, we isolated R1DC41T, a novel member of the Bacillaceae family in the Firmicutes phylum. It showed 16S rRNA gene similarity of 94.59-97.36% with closest relatives of Rossellomorea (which was formerly in the Bacillus genus), Domibacillus, Bacillus, and Jeotgalibacillus genera. Based on the multilocus sequence analysis (MLSA), R1DC41T strain formed a separated branch from the listed genera, representing a novel species of a new genus for which the name Mangrovibacillus cuniculi gen. nov., sp. nov. is proposed. Genomic, morphological, and physiological characterizations revealed that R1DC41T is an aerobic, Gram-stain-variable, rod-shaped, non-motile, endospore-forming bacterium. A reduced genome and the presence of numerous transporters used to import the components necessary for its growth and resistance to the stresses imposed by the oligotrophic and salty mangrove sediments make R1DC41T extremely adapted to its environment of origin and to the competitive conditions present within.

Bioturbation Intensity Modifies the Sediment Microbiome and Biochemistry and Supports Plant Growth in an Arid Mangrove System.

In intertidal systems, the type and role of interactions among sediment microorganisms, animals, plants and abiotic factors are complex and not well understood. Such interactions are known to promote nutrient provision and cycling, and their dynamics and relationships may be of particular importance in arid microtidal systems characterized by minimal nutrient input. Focusing on an arid mangrove ecosystem on the central Red Sea coast, we investigated the effect of crab bioturbation intensity (comparing natural and manipulated high levels of bioturbation intensity) on biogeochemistry and bacterial communities of mangrove sediments, and on growth performance of Avicennia marina, over a period of 16 months. Along with pronounced seasonal patterns with harsh summer conditions, in which high sediment salinity, sulfate and temperature, and absence of tidal flooding occur, sediment bacterial diversity and composition, sediment physicochemical conditions, and plant performance were significantly affected by crab bioturbation intensity. For instance, bioturbation intensity influenced components of nitrogen, carbon, and phosphate cycling, bacterial relative abundance (i.e., Bacteroidia, Proteobacteria and Rhodothermi) and their predicted functionality (i.e., chemoheterotrophy), likely resulting from enhanced metabolic activity of aerobic bacteria. The complex interactions among bacteria, animals, and sediment chemistry in this arid mangrove positively impact plant growth. We show that a comprehensive approach targeting multiple biological levels provides useful information on the ecological status of mangrove forests. IMPORTANCE Bioturbation is one of the most important processes that governs sediment biocenosis in intertidal systems. By facilitating oxygen penetration into anoxic layers, bioturbation alters the overall sediment biogeochemistry. Here, we investigate how high crab bioturbation intensity modifies the mangrove sediment bacterial community, which is the second largest component of mangrove sediment biomass and plays a significant role in major biogeochemical processes. We show that the increase in crab bioturbation intensity, by ameliorating the anoxic condition of mangrove sediment and promoting sediment bacterial diversity in favor of a beneficial bacterial microbiome, improves mangrove tree growth in arid environments. These findings have significant implications because they show how crabs, by farming the mangrove sediment, can enhance the overall capacity of the system to sustain mangrove growth, fighting climate change.

Tumor Growth in the High Frequency Medulloblastoma Mouse Model Ptch1+/-/Tis21KO Has a Specific Activation Signature of the PI3K/AKT/mTOR Pathway and Is Counteracted by the PI3K Inhibitor MEN1611.

We have previously generated a mouse model (Ptch1+/-/Tis21KO ), which displays high frequency spontaneous medulloblastoma, a pediatric tumor of the cerebellum. Early postnatal cerebellar granule cell precursors (GCPs) of this model show, in consequence of the deletion of Tis21, a defect of the Cxcl3-dependent migration. We asked whether this migration defect, which forces GCPs to remain in the proliferative area at the cerebellar surface, would be the only inducer of their high frequency transformation. In this report we show, by further bioinformatic analysis of our microarray data of Ptch1+/-/Tis21KO GCPs, that, in addition to the migration defect, they show activation of the PI3K/AKT/mTOR pathway, as the mRNA levels of several activators of this pathway (e.g., Lars, Rraga, Dgkq, Pdgfd) are up-regulated, while some inhibitors (e.g. Smg1) are down-regulated. No such change is observed in the Ptch1+/- or Tis21KO background alone, indicating a peculiar synergy between these two genotypes. Thus we investigated, by mRNA and protein analysis, the role of PI3K/AKT/mTOR signaling in MBs and in nodules from primary Ptch1+/-/Tis21KO MB allografted in the flanks of immunosuppressed mice. Activation of the PI3K/AKT/mTOR pathway is seen in full-blown Ptch1+/-/Tis21KO MBs, relative to Ptch1+/-/Tis21WT MBs. In Ptch1+/-/Tis21KO MBs we observe that the proliferation of neoplastic GCPs increases while apoptosis decreases, in parallel with hyper-phosphorylation of the mTOR target S6, and, to a lower extent, of AKT. In nodules derived from primary Ptch1+/-/Tis21KO MBs, treatment with MEN1611, a novel PI3K inhibitor, causes a dramatic reduction of tumor growth, inhibiting proliferation and, conversely, increasing apoptosis, also of tumor CD15+ stem cells, responsible for long-term relapses. Additionally, the phosphorylation of AKT, S6 and 4EBP1 was significantly inhibited, indicating inactivation of the PI3K/AKT/mTOR pathway. Thus, PI3K/AKT/mTOR pathway activation contributes to Ptch1+/-/Tis21KO MB development and to high frequency tumorigenesis, observed when the Tis21 gene is down-regulated. MEN1611 could provide a promising therapy for MB, especially for patient with down-regulation of Btg2 (human ortholog of the murine Tis21 gene), which is frequently deregulated in Shh-type MBs.

Fine-scale metabolic discontinuity in a stratified prokaryote microbiome of a Red Sea deep halocline.

Deep-sea hypersaline anoxic basins are polyextreme environments in the ocean's interior characterized by the high density of brines that prevents mixing with the overlaying seawater, generating sharp chemoclines and redoxclines up to tens of meters thick that host a high concentration of microbial communities. Yet, a fundamental understanding of how such pycnoclines shape microbial life and the associated biogeochemical processes at a fine scale, remains elusive. Here, we applied high-precision sampling of the brine-seawater transition interface in the Suakin Deep, located at 2770 m in the central Red Sea, to reveal previously undocumented fine-scale community structuring and succession of metabolic groups along a salinity gradient only 1 m thick. Metagenomic profiling at a 10-cm-scale resolution highlighted spatial organization of key metabolic pathways and corresponding microbial functional units, emphasizing the prominent role and significance of salinity and oxygen in shaping their ecology. Nitrogen cycling processes are especially affected by the redoxcline with ammonia oxidation processes being taxa and layers specific, highlighting also the presence of novel microorganisms, such as novel Thaumarchaeota and anammox, adapted to the changing conditions of the chemocline. The findings render the transition zone as a critical niche for nitrogen cycling, with complementary metabolic networks, in turn underscoring the biogeochemical complexity of deep-sea brines.

Discovery of Afifi, the shallowest and southernmost brine pool reported in the Red Sea.

The previously uncharted Afifi brine pool was discovered in the eastern shelf of the southern Red Sea. It is the shallowest brine basin yet reported in the Red Sea (depth range: 353.0 to 400.5 m). It presents a highly saline (228 g/L), thalassohaline, cold (23.3 °C), anoxic brine, inhabited by the bacterial classes KB1, Bacteroidia and Clostridia and the archaeal classes Methanobacteria and Deep Sea Euryarcheota Group. Functional assignments deduced from the taxonomy indicate methanogenesis and sulfur respiration to be important metabolic processes in this environment. The Afifi brine was remarkably enriched in dissolved inorganic carbon due to microbial respiration and in dissolved nitrogen, derived from anammox processes and denitrification, according to high δ15N values (+6.88‰, AIR). The Afifi brine show a linear increase in δ18O and δD relative to seawater that differs from the others Red Sea brine pools, indicating a non-hydrothermal origin, compatible with enrichment in evaporitic environments. Afifi brine was probably formed by venting of fossil connate waters from the evaporitic sediments beneath the seafloor, with a possible contribution from the dehydration of gypsum to anhydrite. Such origin is unique among the known Red Sea brine pools.

Autogenous regulation of Escherichia coli polynucleotide phosphorylase expression revisited.

The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5' end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5' double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5' end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5' fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.

Consistent bacterial selection by date palm root system across heterogeneous desert oasis agroecosystems.

Highly productive conventional agroecosystems are spatially embedded in resource-homogeneous systems and count on generally nutrient-rich soils. On the contrary, desert oases are isolated, the soil is relatively poor, but yet productivity is similar to conventional agroecosystems. Soil dominates over plant as the main factor shaping root-associated microbiomes in conventional agroecosystems. We hypothesize that in desert oasis, the environmental discontinuity, the resource paucity and limited microbial diversity of the soil make the plant a prevailing factor. We have examined the bacterial communities in the root system of date palm (Phoenix dactylifera), the iconic keystone species of the oases, grown in heterogeneous soils across a broad geographic range (22,200 km2 surface area) of the Sahara Desert in Tunisia. We showed that, regardless of the edaphic conditions and geographic location, the plant invariably selects similar Gammaproteobacteria- and Alphaproteobacteria-dominated bacterial communities. The phylogeny, networking properties and predicted functionalities of the bacterial communities indicate that these two phyla are performing the ecological services of biopromotion and biofertilization. We conclude that in a desert agroecosystem, regardless of the soil microbial diversity baseline, the plant, rather than soil type, is responsible of the bacterial community assembly in its root systems, reversing the pattern observed in conventional agroecosystem.

The role of fungi in heterogeneous sediment microbial networks.

While prokaryote community diversity and function have been extensively studied in soils and sediments, the functional role of fungi, despite their huge diversity, is widely unexplored. Several studies have, nonetheless, revealed the importance of fungi in provisioning services to prokaryote communities. Here, we hypothesise that the fungal community plays a key role in coordinating entire microbial communities by controlling the structure of functional networks in sediment. We selected a sediment environment with high niche diversity due to prevalent macrofaunal bioturbation, namely intertidal mangrove sediment, and explored the assembly of bacteria, archaea and fungi in different sediment niches, which we characterised by biogeochemical analysis, around the burrow of a herbivorous crab. We detected a high level of heterogeneity in sediment biogeochemical conditions, and diverse niches harboured distinct communities of bacteria, fungi and archaea. Saprotrophic fungi were a pivotal component of microbial networks throughout and we invariably found fungi to act as keystone species in all the examined niches and possibly acting synergistically with other environmental variables to determine the overall microbial community structure. In consideration of the importance of microbial-based nutrient cycling on overall sediment ecosystem functioning, we underline that the fungal microbiome and its role in the functional interactome cannot be overlooked.