Cultured meat in the European Union: Legislative context and food safety issues.

The current food system, which is responsible for about one third of all global gas emissions, is considered one of the main causes of resource depletion. For this reason, scientific research is investigating new alternatives capable of feeding an ever-growing population that is set to reach 9-11 billion by 2050. Among these, cell-based meat, also called cultured meat, is one possible solution. It is part of a larger branch of science called cellular agriculture, whose goal is to produce food from individual cells rather than whole organisms, tracing their molecular profile. To date, however, cultured meat aroused conflicting opinions. For this reason, the aim of this review was to take an in-depth look at the current European legislative framework, which reflects a 'precautionary approach' based on the assumption that these innovative foods require careful risk assessment to safeguard consumer health. In this context, the assessment of possible risks made it possible not only to identify the main critical points during each stage of the production chain (proliferation, differentiation, scaffolding, maturation and marketing), but also to identify solutions in accordance with the recommendations of the European Food Safety Authority (EFSA). Further, the main challenges related to organoleptic and nutritional properties have been reviewed.. Finally, possible future markets were studied, which would complement that of traditional meat, implementing the offer for the consumer, who is still sceptical about the acceptance of this new product. Although further investigation is needed, the growing demand for market diversification and the food security opportunities associated with food shortages, as well as justifying the commercialisation of cultured meat, would present an opportunity to position cultured meat as beneficial.

Catatonia in systemic lupus erythematosus: case based review.

Catatonia is a rare psychomotor syndrome characterized by stupor, posturing and echophenomena. It can be associated with schizophrenia, infections, drugs and autoimmune causes like anti N-methyl D-aspartate (NMDA) receptor encephalitis and systemic lupus erythematosus (SLE). Here we report two cases of systemic lupus erythematosus with catatonia, who improved with immunosuppressive treatment and review the cases described in the literature. The first case presented with fever, pancytopenia, toxic epidermal necrolysis (TEN)-like rash and later developed catatonia and macrophage activation syndrome (MAS). The second case presented with acute cutaneous lupus erythematosus (ACLE), fever, alopecia, polyarthralgias, nephritis, cytopenias along with catatonia. Successful management of this syndrome requires prompt recognition and treatment with immunosuppression as well as benzodiazepines with or without electroconvulsive therapy (ECT).

Teleconsultation experience with the idiopathic inflammatory myopathies: a prospective observational cohort study during the COVID-19 pandemic.

Teleconsultation has assumed a central role in the management of chronic and disabling rheumatic diseases, such as the idiopathic inflammatory myopathies (IIM), during COVID-19. However, the feasibility, challenges encountered, and outcomes remain largely unexplored. Here, we describe our teleconsultation experience in a prospectively followed cohort of adult and juvenile IIM. 250 IIM enrolled into the MyoCite cohort (2017-ongoing) were offered the option of audio/visual teleconsultation using WhatsApp during the nationwide lockdown. Clinical outcomes (major/minor relapse) and prescription changes were compared between IIM subsets. Socio-demographic and clinico-serological characteristics of those who sought teleconsultation were compared with those who did not. 151 teleconsultations were sought over a 93 day period by 71 (52.2%) of 136 IIM (median age 38 years, F:M 4.5:1). Nearly one-third (38%) consulted on an emergency basis, with voice consultations being the primary medium of communication. Over a quarter (26.8%) reported relapse (15.5% minor, 11.3% major), these being more common in JDM [71.4%, OR 8.9 (1.5-51)] as compared with adult IIM, but similar across various antibody-based IIM subtypes. Patients who relapsed required more consultations [2(2-3) vs 1(1-2), p 0.009]. The demographic and socioeconomic profile of the patients seeking consultation (n = 71) was not different from those who did not (n = 65). Voice-based teleconsultations may be useful to diagnose and manage relapses in IIM during the pandemic. Patient education for meticulous and timely reporting may be improve care, and larger multicentre studies may identify subsets of IIM that require greater care and early tele-triage for effective management of the condition.

A prospective study of novel disease activity indices for ankylosing spondylitis.

There is an ongoing quest for robust disease activity measures in Ankylosing spondylitis (AS). Thus, we prospectively validated two new disease activity indices, Simplified AS Disease Activity Score (SASDAS) and modified Juvenile Spondyloarthritis Disease Activity Score (JSpADA). Patients with AS were assessed for BASDAI, ASDAS and other outcome measures at baseline and 3 months. Comparisons were drawn between those with juvenile onset, early disease and peripheral involvement, with the rest. Fisher's r to Z transformation was used to compare correlations. Receiver-operating characteristic (ROC) curves were used to calculate cutoffs for inactive, low, high and very high disease activity. Of the 107 patients (mean age-29 years) of 6-years long disease, 38.3% had a juvenile onset. SASDAS and modified JSpADA exhibited excellent correlation with BASDAI and ASDAS (all p < 0.001) and were higher in active vs. inactive disease. Treatment responders had a greater fall in SASDAS and modified JSpADA as compared to non-responders. The novel scores were higher in those with peripheral disease. Only SASDAS could discriminate early from late disease. Based on the previously proposed cutoffs, optimal scores for inactive, moderate, high and very high disease activity were deduced. SASDAS-CRP showed better internal consistency than SASDAS-ESR and correlated better with ASDAS-CRP in late disease (Z = 3.04; p = 002) and those with adult onset disease (Z = 2.18; p = 0.03). SASDAS and Modified JSpADA perform as well as standard complex scores and have potential for simpler daily use. From our analyses, SASDAS with CRP performs better than SASDAS-ESR, pending further validation.

Microsporidial myositis in adult-onset immunodeficiency: case-based review.

Polymyositis is a diagnosis of exclusion. In patients with odd features, it can be of infective etiology. A high index of suspicion is required for diagnosis. A 55-year-old gentleman presented with gradual-onset proximal muscle weakness. Examination revealed mild distal weakness but no rash. Muscle enzymes were raised and tests for autoantibodies were negative. Biopsy revealed microsporidiosis. In view of this unusual infection, immunodeficiency was considered and he was found to have lymphopenia which antedated his illness. Later, he developed cranial nerve palsies due to multiple lesions in the pons. In addition, he had Cytomegalovirus viremia. Literature was reviewed to identify 20 cases of microsporidial myositis, its presentation, underlying immunodeficient state, and clinical course. Infective polymyositis should be considered in a patient with paucity of clinical and serological autoimmune features. Lymphopenia can point to underlying immunodeficiency. CMV infection could be the contributor to or bystander-effect of idiopathic lymphopenia.

Forward versus inverse planning in oropharyngeal cancer: A comparative study using physical and biological indices.

CONTEXT: Possible benefits of inverse planning. AIMS: To analyze possible benefits of inverse planning intensity modulated radiation therapy (IMRT) over field-in-field 3D conformal radiation therapy (FIF-3DCRT) and to evaluate the differences if any, between low (6 Million Volts) and high energy (15 Million Volts) IMRT plans. MATERIALS AND METHODS: Ten patients with squamous cell carcinoma of oropharynx, previously treated with 6 MV step and shoot IMRT were studied. V100, V33, V66 , mean dose and normal tissue complication probabilities (NTCP) were evaluated for parotid glands. Maximum dose and NTCP were the parameters for spinal cord. STATISTICAL ANALYSIS USED: A two-tailed t-test was applied to analyze statistical significance between the different techniques. RESULTS: For combined parotid gland, a reduction of 4.374 Gy, 9.343 Gy and 7.883 Gy were achieved for D100, D66 and D33, respectively in 6 MV-IMRT when compared with FIF-3DCRT. Spinal cord sparing was better in 6 MV-IMRT (40.963 ± 2.650), with an average reduction of maximum spinal cord dose by 7.355 Gy from that using the FIF-3DCRT technique. The uncomplicated tumor control probabilities values were higher in IMRT plans thus leading to a possibility of dose escalation. CONCLUSIONS: Though low-energy IMRT is the preferred choice for treatment of oropharyngeal cancers, FIF-3DCRT must be given due consideration as a second choice for its well established advantages over traditional conventioan technique.

Dosimetric comparison between bone marrow sparing intensity-modulated radiation therapy and conventional techniques in the treatment of cervical cancer: a retrospective study.

PURPOSE: To investigate enlargement of prostate volume by edema during brachytherapy seed implantation and develop a nomogram model to calculate air-kerma strength (AKS) required for implantation of the enlarged transient prostatic volume. MATERIALS AND METHODS: The prostate volume was measured prior and after seed implantation using trans-rectal ultrasound imaging in the operating room to obtain volume enlargement. A nomogram model was developed that calculates AKS required for implantation of the enlarged transient prostate volume with optimal dose coverage. RESULTS: The measured prostate enlargement in this study was up to 60% of the initial volume. The effective prostatic volume enlargement was calculated for three isotopes: 125I, 103Pd and 131Cs. The effective volume enlargement for 125I implants was relatively small (< 10%) because of its long half-life. For 103Pd and 131Cs with short half-lives, additional AKS up to 20% and 30%, respectively, might be required to provide appropriate dose coverage of possible enlarged prostatic volumes. CONCLUSIONS: Prostate volume enlargement should be considered to obtain optimal dose coverage particularly for short half-life isotopes such as 131Cs and 103Pd. The nomogram model developed in this work provides the AKS required for implants with a wide range of prostatic volume enlargements (5-100%) for three isotopes. KEYWORDS: prostate brachytherapy, nomogram, airkerma strength, edema, volume enlargement.

Evaluation of safety profile of black shilajit after 91 days repeated administration in rats.

OBJECTIVE: To evaluate the safety of shilajit by 91 days repeated administration in different dose levels in rats. METHODS: In this study the albino rats were divided into four groups. Group I received vehicle and group II, III and IV received 500, 2 500 and 5 000 mg/kg of shilajit, respectively. Finally animals were sacrificed and subjected to histopathology and iron was estimated by flame atomic absorption spectroscopy and graphite furnace. RESULTS: The result showed that there were no significant changes in iron level of treated groups when compared with control except liver (5 000 mg/kg) and histological slides of all organs revealed normal except negligible changes in liver and intestine with the highest dose of shilajit. The weight of all organs was normal when compared with control. CONCLUSIONS: The result suggests that black shilajit, an Ayurvedic formulation, is safe for long term use as a dietary supplement for a number of disorders like iron deficiency anaemia.

Anti Diabetic and Hypolipidemic Activity of Bark of Ethanolic Extract of Ougeinia Oojeinensis (ROXB.).

The hypoglycemic and hypolipidemic effect of Ethanolic extract of Ougeinia oojeinensis (200mg/kg) bark was evaluated with measurements including, Body weight, blood glucose level, urine glucose and biochemical parameters. The ethanolic extracts of the powdered bark was tested for its efficacy in alloxan-induced diabetic rats. Animals were induced for diabetes with Alloxan (150 mg/kg of body weight- i.p.) and treated orally with Ethanolic extract of Ougeinia oojeinensis. The extracts were also evaluated for acute oral toxicity studies and its effect on different biochemical parameters. The extracts showed significant (p<0.01) antihyperglycemic and hypolipidemic activity as compared to diabetic control. The extract shows beneficial effects on blood glucose and urine glucose level. It also reduces the elevated biochemical parameters such as triglycerides (TGL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), Total Cholesterol (TC) and increased the reduced level of high density lipoprotein (HDL) and body weight, which might be due to presence of steroids, tannins, alkaloids and triterpenoids present in that extract. Thus ethanolic extract could serve as good oral hypoglycemic agents and seems to be promising for the development of phytomedicines for diabetes mellitus.

Tetrameric malate dehydrogenase from a thermophilic Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and characterization of the recombinant enzyme.

The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid. The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa. Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species. Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs. MDHs and LDHs resemble each other in catalytic mechanism and several other respects. However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs. The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric. A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E. coli plasmid vector. BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E. coli strain carrying the clone. MDH purified from BI and that purified from the E. coli strain carrying the MDH gene clone appear to be identical proteins by several criteria. A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters. Whereas the MDH is a stable tetramer in the pH range 5-7, it appears to be converted into a stable dimer at pH 3.5. This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH.

Catalytic-rate improvement of a thermostable malate dehydrogenase by a subtle alteration in cofactor binding.

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2'- and 3'-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.

Overexpression of the Thermus aquaticus B malate dehydrogenase-encoding gene in Escherichia coli.

Expression of the Thermus aquaticus B malate dehydrogenase (MDH)-encoding gene (mdh), cloned in Escherichia coli, was initially at a relatively low level (0.1% of soluble cell protein) and was effected by read-through from the tac promoter in the plasmid vector used. An enhancement in expression to 0.4% of soluble cell protein was achieved by shortening the intervening sequence between the promoter and the translation start codon of mdh. An NdeI restriction site (5'-CAT-ATG-3') was engineered in the shortened fragment, which also changed the start codon from GTG to ATG. This resulted in an eightfold increase in expression, to 3.2% of soluble cell protein. Expression was further increased by subcloning the mdh gene via the engineered NdeI site, into two plasmid expression vectors, one carrying the E. coli trpP promoter and the other the E. coli mdhP promoter. In both these expression systems, 40-50% of the soluble cell protein was T. aquaticus MDH. This suggests that expression of the cloned T. aquaticus mdh in E. coli is enhanced predominantly by the optimisation of transcription and translation initiation signals. Moreover, the base composition of the coding region and the pattern of codon usage dictated by it appear to have little effect on expression. Heat treatment of the cell extract at 85 degrees C further effected purification of T. aquaticus MDH to over 80% of the soluble cell protein. The MDHs purified to homogeneity from the high-expression clones were identical with the MDH isolated from T. aquaticus B cells with respect to all measured parameters.

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B.

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.

Regulation of isocitrate dehydrogenase by phosphorylation in Escherichia coli K-12 and a simple method for determining the amount of inactive phosphoenzyme.

In several Escherichia coli K-12 strains grown on a limiting concentration of glucose, isocitrate dehydrogenase (IDH) was inactivated about 90% after cessation of growth upon exhaustion of the glucose. Such inactivation has been previously observed in several E. coli strains but not in E. coli K-12 (unless acetate was added to the bacterial culture when growth ceased). IDH was inactivated 75 to 80% in all E. coli K-12 strains we examined during growth on acetate. The inactivation involved phosphorylation of the enzyme and is considered to be a regulatory mechanism facilitating metabolite flow along the glyoxylate shunt. Phospho-IDH interacted with antibodies to enzymatically active IDH. We have devised a method, based on this immunological cross-reaction, for determining the proportions of active and inactive (phospho-) IDH in cell extracts.

Isocitrate dehydrogenase from thermophilic and mesophilic bacteria. Isolation and some characteristics.

1. Simple methods incorporating the principle of selective enzyme elution from a triazinyl dye adsorbent with a mixture of NADP+ and isocitrate are described for isolating NADP+-linked isocitrate dehydrogenase in pure state from several mesophilic and thermophilic bacteria. 2. Several characteristics of the isocitrate dehydrogenases have been examined, viz. molecular size, amino acid composition including the content of sulphydryl groups, thermostability and structural homology by the criterion of immunological cross-section.

Stability and immunological cross-reactivity of malate dehydrogenases from mesophilic and thermophilic sources.

The thermostability in vitro of dimeric and tetrameric malate dehydrogenases [S)-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mesophilic and thermophilic bacteria shows a good correlation to the growth temperature of the source organism but no consistent relationship to enzyme subunit structure. The thermophile malate dehydrogenases are, in general, more resistant to the surfactants, sodium dodecyl sulphate (SDS) and hexadecyltrimethylammonium bromide, and to the denaturants, guanidinium chloride and urea, than their mesophilic counterparts, with the dimer in each thermal class being more resistant to the chemical perturbants than the tetramer. Sedimentation analysis suggests that denaturation of the malate dehydrogenases by acid-periodate or SDS produces discrete subunits, whereas denaturation by guanidinium chloride followed by carboxymethylation yields ill-defined protein species. SDS and acid-periodate were therefore preferred to generate denatured malate dehydrogenases for use as immunogens and antigens. The native malate dehydrogenases exhibit immunological cross-reactivity only when they are in the same oligomeric form and derived from closely related species, which may, however, be from different thermal classes. Taking immunological cross-reactivity as an indicator of structural similarity, this supports the idea that the thermophilic trait evolved independently within each phyletic line. With denatured malate dehydrogenases as immunogens and antigens, cross-reactivity is manifested between all the malate dehydrogenases examined. This suggests that appreciable primary structural homology exists between the malate dehydrogenases, whether dimeric or tetrameric, from thermophiles and mesophiles and from various taxa.

Action of surfactants on porcine heart malate dehydrogenase isoenzymes and a simple method for the differential assay of these isoenzymes.

The cationic surfactant, cetyl (hexadecyl) trimethylammonium bromide (CTAB), completely inactivates porcine heart cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) at concentrations (of surfactant) which do not affect the activity of the mitochondrial isoenzyme. These concentrations are close to, or higher than, the critical micelle concentration of CTAB. An increase in the ionic strength of the medium significantly retards the CTAB-induced inactivation of the cytoplasmic enzyme. The enzyme is also markedly protected against CTAB inactivation by NADH; L-malate on its own has no effect but a combination of NADH and L-malate affords greater protection than NADH alone. The CTAB inactivation is not reversed by dilution of the surfactant. The highly selective action of CTAB on the two malate dehydrogenases, which correlates well with their electrostatic charges, has been exploited for a simple and reliable differential assay of these isoenzymes. The anionic surfactant, sodium dodecyl sulphate (SDS), at concentrations well below the critical micelle concentration, inactivates both isoenzymes, but the mitochondrial enzyme is significantly more sensitive than its cytoplasmic counterpart. There is thus some correlation, though not as strong as with CTAB, between SDS inactivation and the charges of the two malate dehydrogenases. An increase in ionic strength has opposite effects on the two isoenzymes: the mitochondrial enzyme becomes more resistant and the cytoplasmic enzyme less so. Both isoenzymes are rendered more resistant to SDS by the inclusion of NADH. Inactivation of the enzymes caused by short exposure to SDS is largely reversed by dilution of the detergent, but longer exposure leads to progressive irreversible loss of activity. NADH very effectively protects the isoenzymes against irreversible inactivation. It is likely that a reversible phase of inactivation precedes an irreversible phase and that in the former phase SDS acts competitively with NADH. Both malate dehydrogenases possess considerable resistance to the nonionic detergent, Triton X-100.