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Androstenedione (AD) is a key intermediate in the body's steroid metabolism, used as a precursor for several steroid substances, such as testosterone, estradiol, ethinyl estradiol, testolactone, progesterone, cortisone, cortisol, prednisone, and prednisolone. The world market for AD and ADD (androstadienedione) exceeds 1000 tons per year, which stimulates the pharmaceutical industry's search for newer and cheaper raw materials to produce steroidal compounds. In light of this interest, we aimed to investigate the progress of AD biosynthesis from phytosterols by prospecting scientific articles (Scopus, Web of Science, and Google Scholar databases) and patents (USPTO database). A wide variety of articles and patents involving AD and phytosterol were found in the last few decades, resulting in 108 relevant articles (from January 2000 to December 2021) and 23 patents of interest (from January 1976 to December 2021). The separation of these documents into macro, meso, and micro categories revealed that most studies (articles) are performed in China (54.8%) and in universities (76%), while patents are mostly granted to United States companies. It also highlights the fact that AD production studies are focused on "process improvement" techniques and on possible modifications of the "microorganism" involved in biosynthesis (64 and 62 documents, respectively). The most-reported "process improvement" technique is "chemical addition" (40%), which means that the addition of solvents, surfactants, cofactors, inducers, ionic liquids, etc., can significantly increase AD production. Microbial genetic modifications stand out in the "microorganism" category because this strategy improves AD yield considerably. These documents also revealed the main aspects of AD and ADD biosynthesis: Mycolicibacterium sp. (basonym: Mycobacterium sp.) (40%) and Mycolicibacterium neoaurum (known previously as Mycobacterium neoaurum ) (32%) are the most recurrent species studied. Microbial incubation temperatures can vary from 29 °C to 37 °C; incubation can last from 72 h to 14 days; the mixture is agitated at 140 to 220 rpm; vegetable oils, mainly soybean, can be used as the source of a mixture of phytosterols. In general, the results obtained in the present technological prospecting study are fundamental to mapping the possibilities of AD biosynthesis process optimization, as well as to identifying emerging technologies and methodologies in this scenario.

Palm oil production chain generates a greasy residue in the refining stage, the Palm Oil Deodorizer Distillate (PODD), mainly composed of free fatty acids. Palm oil is also used industrially to fry foods, generating a residual frying oil (RFO). In this paper, we aimed to produce lipase from palm agro-industrial wastes using an unconventional yeast. RFO_palm, from a known source, consisted of 0.11% MAG + FFA, 1.5% DAG, and 97.5 TAG, while RFO_commercial, from a commercial restaurant, contained 6.7% of DAG and 93.3% of TAG. All palm oil wastes were useful for extracellular lipase production, especially RFO_commercial that provided the highest activity (4.9 U/mL) and productivity (465 U/L.h) in 75 h of processing time. In 48 h of process, PODD presented 2.3 U/mL of lipase activity and 48.5 U/L.h of productivity. RFO_commercial also showed the highest values for lipase associated to cell debris (843 U/g). This naturally immobilized biocatalyst was tested on hydrolysis reactions to produce Lipolyzed Milk Fat and was quite efficient, with a hydrolysis yield of 13.1% and 3-cycle reuse. Therefore, oily palm residues seem a promising alternative to produce lipases by the non-pathogenic yeast  Y. lipolytica and show great potential for industrial applications., Competing Interests: Conflict of interestAll authors declare no competing/ conflicts of interests. The funders had no decision on the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results., (© King Abdulaziz City for Science and Technology 2021.)

The lipolytic yeast Yarrowia lipolytica produces cell-wall-associated lipases, namely Lip7p and Lip8p, that could have interesting properties as catalyst either in free (released lipase fraction-RLF) or cell-associated (cell-bound lipase fraction-CBLF) forms. Herein, a mixture of waste soybean frying oil, yeast extract and bactopeptone was found to favor the enzyme production. Best parameters for lipase activation and release from the cell wall by means of acoustic wave treatment were defined as: 26 W/cm 2 for 1 min for CBLF and 52 W/cm 2 for 2 min for RLF. Optimal pH and temperature values for lipase activity together with storage conditions were similar for both the free enzyme and cell-associated one: pH 7.0; T = 37 °C; and > 70% residual activity for 60 days at 4, - 4 °C and for 15 days at 30 °C.

Lipase activity (337 U/g dry weight of cell debris) was detected in cell debris after ultrasound treatment of Yarrowia lipolytica cells cultivated in residual frying palm oil. It is a naturally immobilized lipase with protein content of 47%, herein called LipImDebri. This immobilized biocatalyst presents low hydrophobicity (8%), that can be increased adjusting pH and buffer type. Despite apparent intact cells, electron microscopy showed a shapeless and flat surface for LipImDebri and optical microscopy revealed no cell viability. Besides, an inferior mean diameter (3.4 mm) in relation to whole cells reveals structure modification. A high negative zeta potential value (- 33.86 mV) for pH 6 and 25 °C suggests that LipImDebri is a stable suspension in aqueous solution. Fourier Transform Infrared Spectra (FTIR) expose differences between LipImDebri and extracellular lipase extract signaling a physical interaction between enzyme and cell debris, which is possibly the reason for the high thermostability ( k d  = 0.246 h -1 ; t 1/2  = 2.82 h at 50 °C, pH 7.0). A good adjustment of LipImDebri kinetic data with Hill equation ( R 2  = 0.95) exposes an allosteric behavior related to the presence of more than one lipase isoform. These features reveal that LipImDebri can be a good catalyst for industrial applications., Competing Interests: Conflict of interestAll authors declare no competing/conflicts of interests. The funders had no decision on the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results., (© King Abdulaziz City for Science and Technology 2020.)

Lipase immobilized on Yarrowia lipolytica cell debris after sonication of yeast cells (LipImDebri) was used in hydrolysis reaction as a novel strategy to produce lipolyzed milk fat (LMF). Extracellular (4732.1 U/L), intracellular (130.0 U/g), and cell debris (181.0 U/g) lipases were obtained in a 4 L bioreactor using residual frying oil as inducer in 24 h fermentation process. LipImDebri showed a good operational stability retaining 70% of lipolytic activity after the second cycle and 40% after the fourth. The highest degree of hydrolysis (28%) was obtained with 500 mg LipImDebri for 6 h of lipolysis of anhydrous milk fat. LMF produced with LipImDebri presented high contents of oleic (35.2%), palmitic (25.0%), and stearic (15.4%) acids and considerable amounts of odor-active short and medium chain fatty acids (C:4⁻C:10) (8.13%).

Background: Piroplasmosis caused by the Babesia microti-like piroplasm (Bml) is increasingly being detected in dogs in Europe. Sick dogs show acute disease with severe anaemia associated with thrombocytopenia with a poor response to current available drugs. This study assesses the safety and tolerance of three treatments and compares their efficacy over a full year of follow up in dogs naturally infected with Bml., Methods: Fifty-nine dogs naturally infected with Bml were randomly assigned to a treatment group: imidocarb dipropionate (5 mg/kg SC, 2 doses 14 d apart) (IMI); atovaquone (13.3 mg/kg PO q 8 h, 10 d)/azithromycin (10 mg/kg PO q 24 h, 10 d) (ATO); or buparvaquone (5 mg/kg IM, 2 d apart)/azithromycin (same dosage) (BUP). Before and after treatment (days 15, 45, 90 and 360), all dogs underwent a physical exam, blood tests and parasite detection (blood cytology and PCR). Clinical efficacy was assessed by grading 24 clinical and 8 clinicopathological signs from low to high severity., Results: Before treatment, most dogs had severe regenerative anaemia (88.13%) and thrombocytopenia (71.4%). On treatment Day 45, clinical signs were mostly reduced in all dogs, and by Day 90, practically all dogs under the ATO or BUP regimen were clinically healthy (76.4 and 88%, respectively). Highest percentage reductions in laboratory abnormalities (82.04%) were detected in animals treated with ATO. Over the year, clinical relapse of Bml was observed in 8 dogs (8/17) treated with IMI. However, on Day 360, these animals had recovered clinically, though clinicopathological abnormalities were still present in some of them. Parasitaemia was PCR-confirmed on Days 90 and 360 in 47.05 and 50% of dogs treated with ATO, 68 and 60.08% with BUP, and 94.1 and 73.3% with IMI, respectively. Even after 360 days, 13.3% of the dogs treated with IMI returned a positive blood cytology result., Conclusions: IMI showed the worse clinical and parasitological, efficacy such that its use to treat Bml infection in dogs is not recommended. The treatments ATO and BUP showed better efficacy, though they were still incapable to completely eliminate PCR-proven infection at the recommended dose. All three treatments showed good tolerance and safety with scarce adverse events observed.

Background: In north-western Spain, piroplamosis caused by Theileria annae is now recognized as a serious problem because veterinarians, despite being aware of the clinical signs of piroplasmosis, lack the necessary information on its epidemiology or specific diagnostic tools for its management. This, along with the fact that T. annae infection is also refractory to current piroplamosis treatments, prompted this study designed to assess the clinical presentation and diagnosis of this largely unknown parasitic disease in dogs., Methods: One hundred and twenty dogs in NW Spain suspected clinically of having piroplasmosis were examined and piroplasm species detected by light microscopy (LM) observation of Giemsa-stained blood smears, immunofluorescent antibody test (IFAT), and PCR plus sequencing., Results: Seventy five of the sick dogs were confirmed to be infected with T. annae by PCR (designated "true infection cases"). Intraerythrocytic ring-shaped bodies morphologically compatible with small piroplasms were observed by LM in 59 (57 true infections) of the 120 blood samples. Anti-Babesia antibodies were detected by IFAT in 59 of the 120 sera (55 of which were "true infections"). Using PCR as the reference method, moderate agreement was observed between positive LM vs PCR and IFAT vs PCR results (kappa values: 0.6680 and 0.6017, respectively). Microscopy examination and IFAT were moderately sensitive in detecting the pathogen (76% and 73.3%, respectively). In the 75 cases of "true infection", the most common clinical signs observed were pale mucous membranes, anorexia and apathy. Blood cell counts consistently revealed severe regenerative anaemia and thrombocytopenia in dogs with piroplasmosis due to T. annae. Young dogs (≤3 year) (p = 0.0001) were more susceptible to the disease., Conclusion: Microscopy showed moderate diagnostic sensitivity for acute T. annae infection while IFAT-determined antibody titres were low (1/64 to 1/128). The infecting species should be therefore confirmed by molecular tests. Our results suggest that the disease affects dogs in regions of Spain bordering the endemic Galicia area where this piroplasm has not been previously reported (Asturias, northern Spain). Further epidemiological surveys based on serological and molecular methods are required to establish the current geographical range of T. annae infection.

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Bone marrow-derived stem cell transplantation is a potentially viable therapeutic option for the treatment of neurodegenerative disease., Material/methods: We have isolated bone marrow stem cells by standard method. We then evaluated the survival of rats' bone marrow mononuclear cells implanted in rats' brain. The cells were extracted from rats' femurs, and marked for monitoring purposes by adenoviral transduction with Green Fluorescent Protein (GFP). Labeled cells were implanted within the area of rats' striatum lesions that were induced a month earlier employing quinolinic acid-based method. The implants were phenotyped by monitoring CD34; CD38; CD45 and CD90 expression. Bone marrow stromal cells were extracted from rats' femurs and cultivated until monolayer bone marrow stromal cells were obtained. The ability of bone marrow stromal cells to express NGF and GDNF was evaluated by RT-PCR., Results: Implanted cells survived for at least one month after transplantation and dispersed from the area of injection towards corpus callosum and brain cortex. Interestingly, passaged rat bone marrow stromal cells expressed NGF and GDNF mRNA., Conclusions: The bone marrow cells could be successfully transplanted to the brain either for the purpose of trans-differentiation, or for the expression of desired growth factors.

Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number-PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site's capacity for anaerobic benzene degradation.

The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHODS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens, Geobacter sulphurreducens, Geothrix fermentans, Shewanella alga, Wolinella succinogenes and 'S. barnesii'. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans, could not. AHODS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes. Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.

Electro-oscillographic recordings show that when rats wake up, behavioral manifestations such as head dorsal extension and snout and eye movements (which occur in this sequence), occasionally followed by brief ear and limb twitches, are preceded by and are simultaneous with theta waves (around 6.5 Hz in frequency) in neocortical areas 3 and 17 and in the hippocampus. Heart rate increases when the animal passes from synchronized sleep to relaxed wakefulness to exploratory behavior. During desynchronized sleep theta waves, similar to those found during alertness, usually precede and are simultaneous with rostrum and eye movements and brief ear and limb twitches. Heart rate also increases during dreaming activity. A high degree of coherence (near 1.0) was found among theta bursts in different hippocampal areas in both highly attentive alertness and dreaming. It is suggested that theta waves are related to attention as part of some kinds of behavior and of the process of dream shaping and expression, both characterized by consciousness.

The electrical activity of the posterior thalamic nuclear complex was studied in rats and related to the simultaneous hippocampal electro-oscillogram. Electrocorticographic tracings and spectral analysis showed that during attentive wakefulness the electro-oscillogram peaked at 8 Hz and in desynchronized sleep it oscillated at regular frequencies between 8 and 9 Hz. In quiet wakefulness, frequency was lower (around 6 Hz) and during synchronized sleep rhythmicity disappeared. The electrophysiological patterns during wakefulness and desynchronized sleep are proposed to be linked to the retrieval and combination of information for the generation of attention and dream content.

Establishing the stationarity and statistical distribution of potentials recorded from the nervous system is crucial for the application of frequency analysis. Both parameters were determined in the electrocorticograms of six adult Wistar rats during wakefulness and desynchronized sleep, during both of which desynchronization prevails. Stationarity of the signals was found to occur during at least 20 s in both states of the wakefulness-sleep cycle. A normal distribution was also found for at least 6.7 s. These findings provide strong support for the use of frequency analysis of brain waves as a reliable method to quantify neural electro-oscillograms.