Formate from THF-C1 metabolism induces the AOX1 promoter in formate dehydrogenase-deficient Komagataella phaffii.

In Komagataella phaffii (Pichia pastoris), formate is a recognized alternative inducer to methanol for expression systems based on the AOX1 promoter (pAOX1). By disrupting the formate dehydrogenase encoding FDH1 gene, we converted such a system into a self-induced one, as adding any inducer in the culture medium is no longer requested for pAOX1 induction. In cells, formate is generated from serine through the THF-C1 metabolism, and it cannot be converted into carbon dioxide in a FdhKO strain. Under non-repressive culture conditions, such as on sorbitol, the intracellular formate generated from the THF-C1 metabolism is sufficient to induce pAOX1 and initiate protein synthesis. This was evidenced for two model proteins, namely intracellular eGFP and secreted CalB lipase from C. antarctica. Similar protein productivities were obtained for a FdhKO strain on sorbitol and a non-disrupted strain on sorbitol-methanol. Considering a K. Phaffii FdhKO strain as a workhorse for recombinant protein synthesis paves the way for the further development of methanol-free processes in K. phaffii.

Viable SARS-CoV-2 Omicron sub-variants isolated from autopsy tissues.

Introduction: Pulmonary and extrapulmonary manifestations have been described after infection with SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). The virus is known to persist in multiple organs due to its tropism for several tissues. However, previous reports were unable to provide definitive information about whether the virus is viable and transmissible. It has been hypothesized that the persisting reservoirs of SARS-CoV-2 in tissues could be one of the multiple potentially overlapping causes of long COVID. Methods: In the present study, we investigated autopsy materials obtained from 21 cadaveric donors with documented first infection or reinfection at the time of death. The cases studied included recipients of different formulations of COVID-19 vaccines. The aim was to find the presence of SARS-CoV-2 in the lungs, heart, liver, kidneys, and intestines. We used two technical approaches: the detection and quantification of viral genomic RNA using RT-qPCR, and virus infectivity using permissive in vitro Vero E6 culture. Results: All tissues analyzed showed the presence of SARS-CoV-2 genomic RNA but at dissimilar levels ranging from 1.01 × 102 copies/mL to 1.14 × 108 copies/mL, even among those cases who had been COVID-19 vaccinated. Importantly, different amounts of replication-competent virus were detected in the culture media from the studied tissues. The highest viral load were measured in the lung (≈1.4 × 106 copies/mL) and heart (≈1.9 × 106 copies/mL) samples. Additionally, based on partial Spike gene sequences, SARS-CoV-2 characterization revealed the presence of multiple Omicron sub-variants exhibiting a high level of nucleotide and amino acid identity among them. Discussion: These findings highlight that SARS-CoV-2 can spread to multiple tissue locations such as the lungs, heart, liver, kidneys, and intestines, both after primary infection and after reinfections with the Omicron variant, contributing to extending knowledge about the pathogenesis of acute infection and understanding the sequelae of clinical manifestations that are observed during post-acute COVID-19.

Role of Dissimilative Pathway of Komagataella phaffii (Pichia pastoris): Formaldehyde Toxicity and Energy Metabolism.

Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO2 through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted.

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production.

Komagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered.

Childhood leukemia and non-Hodgkin's lymphoma survivors lack knowledge about their past disease and possible late effects. I-BFM ELTEC study with Chilean and Croatian participation.

PURPOSE: Late effects in childhood cancer survivors are a major cause of morbidity and mortality. The objective was to establish knowledge about the disease, late effects, self-care practices, application of health knowledge/education, sources of information, and biopsychosocial impact of cancer, and compare the results of Chile and Croatia. METHODS: One-hundred-and-seventy-one, 5-year survivors who were treated for leukemia or non-Hodgkin's lymphoma responded to a questionnaire (119 in Chile and 52 in Croatia). The questionnaire was reviewed by BFM-ELTEC. RESULTS: Health knowledge about past diagnosis and general treatment had 96% Chilean and 85% Croatian survivors. Ninety percent of Chilean and 73% of Croatian survivors were unaware of possible late effects, and half did not know which specialist to visit for follow-up. Forty-six percent of Chilean and 35% of Croatian survivors knew about healthy lifestyles, but most did not practice them. The 74% of Chileans and 87% of Croatian survivors recalled having received health education during treatment. About 50% of survivors in both groups were afraid or anguish, but it was also a growth experience for 60% of Chilean and 42% of Croatian survivors. Eighty-seven percent Chilean and 77% Croatian survivors considered themselves physically independent, while 76% and 75% felt psychologically independent, respectively. CONCLUSION: A significant lack of knowledge about the specific treatment, late effects, and future health in both countries was detected. They did not achieve significant learning with the education received. Psychological sequelae were found that are important to prevent.

Telephone-assisted, parent-mediated CBT for rural Latino youth with anxiety: A feasibility trial.

OBJECTIVE: In this study, we tailor a child anxiety cognitive-behavior therapy (CBT) program to fit the needs of rural Latino/a Spanish-speaking families and examine the feasibility, acceptability, tolerability, and safety of this intervention using 2 modes of service delivery. METHOD: Children (n = 31; age 8-13) with anxiety disorders were recruited from primary care settings and randomized to 1 of 2 modes of parent-mediated CBT bibliotherapy: (1) telephone-delivered, therapist-assisted bibliotherapy (TTB; n = 15), and (2) a more minimal contact, self-directed, bibliotherapy condition (SB; n = 16). Independent evaluators administered a diagnostic interview at baseline and posttreatment; demographic and engagement-related questionnaires were also administered. RESULTS: In the TTB group, 10 of 15 parents (66.7%) completed the requisite number of therapist-assisted bibliotherapy sessions, and in the SB group, 4 of 16 parents (25%) completed the requisite number of self-directed bibliotherapy modules; this comparison was significant, χ2(1) = 5.43, p = .02. In the TTB condition, treatment length and session duration were longer than reported in previous studies. Barriers that were most strongly endorsed were stressors and obstacles competing with treatment, as well as treatment demandingness. Findings revealed good satisfaction across the conditions, although ratings were significantly higher in the TTB group, F = 5.67, p = .028. Remission rates (i.e., no anxiety disorder) for those that provided posttreatment data (N = 25) were 50% and 36% for the TTB and SB groups, respectively, χ2(1) = .465, p = .50. CONCLUSIONS: Implications of these feasibility findings and suggestions for future research are discussed. (clinicaltrials.gov unique identifier: NCT01491880). (PsycINFO Database Record

Randomized Trial of the Parent And Caregiver Active Participation Toolkit for Child Mental Health Treatment.

The purpose of this pilot study was to examine preliminary feasibility, acceptability, and effectiveness of a toolkit (Parent And Caregiver Active Participation Toolkit) to increase parent participation in community-based child mental health services. Study participants included 29 therapists (93% female; M age = 34.1 years; 38% Latino) and 20 parent/child dyads (children 80% female; M age = 8.6 years; parents 40% Latino) in 6 diverse community mental health clinics. Therapists were randomly assigned to standard care or the toolkit with standard care. Therapist and parent survey data and observational coding of treatment sessions were utilized. Mean comparisons and repeated measures analyses were used to test differences between study conditions over 4 months. Results supported preliminary feasibility and acceptability of the toolkit, with therapists assigned to the toolkit participating in ongoing training, adhering to toolkit use, and perceiving the toolkit as feasible and acceptable within their setting. Results preliminarily demonstrated improvement in therapists' job attitudes, as well as actual use of parent engagement strategies. Results also preliminarily demonstrated increases in parent participation in child therapy sessions and more regular attendance, as well as some indication of support for perceived treatment effectiveness. Overall, results suggest the feasibility, acceptability, and potential effectiveness of the toolkit to enhance therapist job attitudes; practices that support parent engagement, parent engagement itself, and consumer perspectives on treatment outcomes; and the potential promise of future research in the area of parent participation interventions in child mental health services.

"Media Addiction" in a 10-Year-Old Boy.

CASE: Bryan is a 10-year-old boy who is brought to his pediatrician by his parents with concerns about oppositional behaviors. Bryan's parents report that he has always been hyperactive and oppositional since a very young age. He has been previously diagnosed with attention-deficit hyperactivity disorder and has been treated with appropriate stimulant medications for several years; however, despite this, his parents feel increasingly unable to manage his difficult behaviors. He refuses to do chores or follow through with household routines. He refuses to go to bed at night. His family feels unable to take him to public places because he "climbs all over everything." At school, he acts up in class, is often disruptive, and requires close supervision by teachers. He was recently kicked off of the school bus. He has very few friends, and his parents state that other children do not enjoy to be around him.Bryan's parents also report that he is "obsessed" with electronics. He spends most his free time watching TV and movies and playing computer games. He has a television in his bedroom because otherwise he "monopolizes" the family television. The family also owns several portable electronic devices that he frequently uses. Bryan insists on watching TV during meals and even that the TV stays on in an adjacent room while showering. He gets up early each morning and turns on the television. He refuses to leave the house unless he can take a portable screen device with him. His parents admit to difficulty placing limits on this behavior because they feel it is the only way to keep his other behaviors under control. His mother explains "it is our only pacifier" and that attempts to place restrictions are met with explosive tantrums and have thus been short lived. These efforts have also been impeded due to the habits of his parents and older sibling, who also enjoy spending a significant amount of time watching television.

Delivering CBT to Rural Latino Children with Anxiety Disorders: A Qualitative Study.

Qualitative methods were used to understand community perspectives about ways to deliver cognitive behavior therapy (CBT) to rural Latino youth with anxiety. First, four focus groups were conducted with 28 bilingual Latino mental health providers to examine perceptions of CBT using telephone based, therapist supported bibliotherapy, and bibliotherapy without therapist support. Second, qualitative interviews were conducted with 15 Latino parents from a rural community to better understand attitudes toward CBT, and modes of service delivery. Qualitative findings revealed that parents were mostly positive about psychotherapy, and the core elements of CBT for anxiety. However, both parents and providers emphasized the need for adaptations to address practical and perceived barriers to treatment, such as time, convenience, homework, and literacy. Many parents spoke favorably of a telephone-based approach that could address many of their perceived barriers, while providers were expressed more negative views. Such findings are important for data-driven treatment development efforts.

"Media addiction" in a 10-year-old boy.

CASE: Bryan is a 10-year-old boy who is brought to his pediatrician by his parents with concerns about oppositional behaviors. Bryan's parents report that he has always been hyperactive and oppositional since a very young age. He has been previously diagnosed with attention-deficit hyperactivity disorder and has been treated with appropriate stimulant medications for several years; however, despite this, his parents feel increasingly unable to manage his difficult behaviors. He refuses to do chores or follow through with household routines. He refuses to go to bed at night. His family feels unable to take him to public places because he "climbs all over everything." At school, he acts up in class, is often disruptive, and requires close supervision by teachers. He was recently kicked off of the school bus. He has very few friends, and his parents state that other children do not enjoy to be around him. Bryan's parents also report that he is "obsessed" with electronics. He spends most his free time watching TV and movies and playing computer games. He has a television in his bedroom because otherwise he "monopolizes" the family television. The family also owns several portable electronic devices that he frequently uses. Bryan insists on watching TV during meals and even that the TV stays on in an adjacent room while showering. He gets up early each morning and turns on the television. He refuses to leave the house unless he can take a portable screen device with him. His parents admit to difficulty placing limits on this behavior because they feel it is the only way to keep his other behaviors under control. His mother explains "it is our only pacifier" and that attempts to place restrictions are met with explosive tantrums and have thus been short lived. These efforts have also been impeded due to the habits of his parents and older sibling, who also enjoy spending a significant amount of time watching television.

Protein measurements of microalgal and cyanobacterial biomass.

The protein content of dry biomass of the microalgae Porphyridium cruentum, Scenedesmus almeriensis, and Muriellopsis sp. and of the cyanobacteria Synechocystis aquatilis and Arthrospira platensis was measured by the Lowry method following disruption of the cells by milling with inert ceramic particles. The measurements were compared with the Kjeldahl method and by elemental analysis. The nitrogen-to-protein conversion factors for biomass obtained from exponentially growing cells with a steady state doubling time of approximately 23 h were 5.95 for nitrogen measured by Kjeldahl and 4.44 for total nitrogen measured by elemental analysis. The protein content in dry biomass ranged from 30% to 55%. The above conversion factors are useful for estimating the protein content of microalgal biomass produced in rapid steady state growth as encountered in many commercial production processes.