Implementing youth participatory action research at a continuation high school.

OBJECTIVE: To describe the process of implementing a Youth Participatory Action Research (YPAR) project at a continuation high school (CHS) and share the results of a youth-designed research project that explores barriers to high school completion. DATA SOURCES AND STUDY SETTING: YPAR was implemented across three cohorts at a CHS in the central coast of California from 2019 to 2022. Student survey respondents were enrolled CHS students between March and April 2021. STUDY DESIGN: A modified YPAR curriculum integrating research methodology and social justice topics was used to guide student-led research that resulted in a cross-sectional survey. DATA COLLECTION: Field notes maintained by the first author documented the process of implementing YPAR including the curriculum, conversations, and research decisions and procedures. A student-designed survey disseminated to all enrolled students resulted in 76 (66%) participant responses. The survey included 18 close-ended questions and three narrative responses. PRINCIPAL FINDINGS: This study details how YPAR methodologies can be translated to a high school credit recovery program. For example, student cohorts were needed to maintain continuity over time. A student-designed survey revealed that 72% of student respondents reported taking care of family members and illuminated high rates of depression symptoms. CONCLUSIONS: This study offers a detailed description of how we implemented YPAR at a credit recovery program and provides student-driven perspectives on educational reform and evaluation. This project addresses the implementation and challenges of using YPAR to engage youth in transformational resistance to rapidly study and improve CHS' policy and practice.

Horizontal gene transfer from genetically modified plants - Regulatory considerations.

Gene technology regulators receive applications seeking permission for the environmental release of genetically modified (GM) plants, many of which possess beneficial traits such as improved production, enhanced nutrition and resistance to drought, pests and diseases. The regulators must assess the risks to human and animal health and to the environment from releasing these GM plants. One such consideration, of many, is the likelihood and potential consequence of the introduced or modified DNA being transferred to other organisms, including people. While such gene transfer is most likely to occur to sexually compatible relatives (vertical gene transfer), horizontal gene transfer (HGT), which is the acquisition of genetic material that has not been inherited from a parent, is also a possibility considered during these assessments. Advances in HGT detection, aided by next generation sequencing, have demonstrated that HGT occurrence may have been previously underestimated. In this review, we provide updated evidence on the likelihood, factors and the barriers for the introduced or modified DNA in GM plants to be horizontally transferred into a variety of recipients. We present the legislation and frameworks the Australian Gene Technology Regulator adheres to with respect to the consideration of risks posed by HGT. Such a perspective may generally be applicable to regulators in other jurisdictions as well as to commercial and research organisations who develop GM plants.

Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences.

Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

Stable Plastid Transformation of Petunia for Studies in Basic Research.

Petunia hybrida is a commercial ornamental plant and is also an important model species for genetic analysis and transgenic research. Here we describe the steps required to isolate stable plastid transformants in P. hybrida using the commercial Pink Wave cultivar. Wave cultivars are popular spreading Petunias sold as ground cover and potted plants. Transgenes introduced into P. hybrida plastids exhibit stable expression over many generations. The development of plastid transformation in P. hybrida provides an enabling technology to bring the benefits of plastid engineering, including maternal inheritance and stable expression of performance-enhancing trait genes, to the important floriculture and horticulture industries.

The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth.

Plant photosynthesis and growth are often limited by the activity of the CO2-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)-a red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, the messenger RNA (mRNA) abundance was ∼25% of rbcL transcript and RsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (∼23% sites active under ambient CO2), the metabolic repair protein RsRca (Rs-activase) was introduced via nuclear transformation. RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m2) and enhanced RsRubisco activation to 75% under elevated CO2 (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producing RsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO2-fixation rates (kcatc) were reduced >95% and CO2/O2 specificity impaired 30-50%. We show differences in hybrid and WT RsRubisco biogenesis in tobacco correlated with assembly in Escherichia coli advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered RsRubisco, an isoform amenable to directed evolution.

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the ßA-ßB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.

Factores de riesgo reproductivo preconcepcional en mujeres de edad fértil del área Este de Guantánamo, 2020 / Preconception reproductive risk factors in women of childbearing age in the East area of Guantanamo in 2020

Introducción: el riesgo reproductivo preconcepcional es la probabilidad que tiene una fémina no gestante de sufrir daño -ella o su producto-, si se involucra en el proceso reproductivo. Objetivo: identificar los factores de riesgo reproductivo preconcepcional en las mujeres en edad fértil del Consultorio Médico de la Familia No. 33, perteneciente al Policlínico Universitario ¨4 de Abril¨, área Este del municipio Guantánamo, en el periodo septiembre de 2019 a marzo de 2020. Método: se realizó un estudio descriptivo con corte transversal, en mujeres en edad fértil (15 a 49 años), pertenecientes a 20 familias del consultorio antes mencionado. El universo estuvo constituido por las 65 mujeres en edad fértil de esas familias. Las variables estudiadas fueron los factores de riesgo socio-laboral-ambiental, riesgos según antecedentes obstétricos desfavorables y riesgos por enfermedades crónicas no transmisibles. Resultados: predominaron las madres solteras, edad menor de 20 años y mayor de 30 años, que representaron el 50,8; 44, 6 y 32,3 %, respectivamente. Los antecedentes obstétricos que más se identificaron fueron el aborto (52,3 %) y la multiparidad (47,7 %). La hipertensión arterial fue la enfermedad crónica no transmisible más encontrada (56,9 %). Conclusiones: todas las mujeres presentan algún tipo de riesgo, lo que coloca a la madre y a su hijo en una situación de vulnerabilidad durante el embarazo

Introduction: preconception reproductive risk is the likelihood that a non-pregnant female will be harmed - herself or her gestation - if she becomes involved in the reproductive process. Objective: to identify the preconception reproductive risk factors in women of childbearing age in the Family Medical Office No. 33, belonging to the ¨4 de Abril¨ University Polyclinic, located in the eastern area of the municipality of Guantánamo, in the period time September 2019 to March 2020. Method: a cross-sectional, descriptive study was conducted on women of childbearing age (15-49 years) from 20 families in the Family Medical Office mentioned before. The universe was made up of the 65 women of childbearing age in those families. The variables studied were social and environmental risk factors, risks according to unfavorable obstetric history and risks for chronic non-communicable diseases. Results: single mothers under 20 and over 30 years old predominated, which represented 50.8%, 44, 6% and 32.3 % respectively. The most common obstetric history identified was abortion (52.3%) and multiparity (47.7%). High blood pressure was the most common chronic non-communicable disease found (56.9%). Conclusions: all women present some kind of risk, aspect that places the mother and child in a vulnerable situation during pregnancy

Neutral, cationic and anionic organonickel and -palladium complexes supported by iminophosphine/phosphinoenaminato ligands.

We report a series of organometallic nickel and palladium complexes containing iminophosphine ligands R2PCH2C(Ph) = N-Dipp (Dipp = 2,6-diisopropylphenyl; R = iPr, La; R = Ph, Lb; and R = o-C6H4OMe, Lc), synthesized by ligand exchange or oxidative addition reactions, and we investigate the capacity of such ligands to undergo reversible deprotonation to the corresponding phosphinoenaminato species. In the attempted ligand exchange reaction of the nickel bis(trimethylsilyl)methyl precursor [Ni(CH2SiMe3)2Py2] with Lb, the iminophosphine acts as a weak acid rather than a neutral ligand, cleaving one of the Ni-C bonds, to afford the phosphinoenaminato complex [Ni(CH2SiMe3)(L'b)(Py)] (L'b = conjugate base of Lb). We disclose a general method for the syntheses of complexes [Ni(CH2SiMe3)(L)(Py)]+ (L = La, Lb or Lc), and demonstrate that iminophosphine deprotonation is a general feature and occurs reversibly in the coordination sphere of the metal. By studying proton exchange reactions of the cation [Ni(CH2SiMe3)(Lb)(Py)]+ with bases of different strength we show that the conjugate phosphinoenaminato ligand in [Ni(CH2SiMe3)(L'b)(Py)] is a base with strength comparable to DBU in THF. The acyl group in the functionalized aryl complex [Ni(p-C6H4COCH3)(Br)(La)] does not interfere in the iminophosphine deprotonation with NaH. The latter reaction affords the unusual anionic hydroxide species [Ni(p-C6H4COCH3)(OH)(L'a)]-Na+, which was isolated and fully characterized.

Rescue of Deletion Mutants to Isolate Plastid Transformants in Higher Plants.

Plastid transformation is an attractive alternative to nuclear transformation enabling manipulation of native plastid genes and the insertion of foreign genes into plastids for applications in agriculture and industrial biotechnology. Transformation is achieved using dominant positive selection markers that confer resistance to antibiotics. The very high copy number of plastid DNA means that a prolonged selection step is required to obtain a uniform population of transgenic plastid genomes. Repair of mutant plastid genes with the corresponding functional allele allows selection based on restoration of the wild type phenotype. The use of deletion rather than point mutants avoids spontaneous reversion back to wild type. Combining antibiotic resistance markers with native plastid genes speeds up the attainment of homoplasmy and allows early transfer of transplastomic lines to soil where antibiotic selection is replaced by selection for photoautotrophic growth. Here we describe our method using the wild type rbcL gene as a plastid transformation marker to restore pigmentation and photosynthesis to a pale green heterotrophic rbcL mutant.

An improved Escherichia coli screen for Rubisco identifies a protein-protein interface that can enhance CO2-fixation kinetics.

An overarching goal of photosynthesis research is to identify how components of the process can be improved to benefit crop productivity, global food security, and renewable energy storage. Improving carbon fixation has mostly focused on enhancing the CO2 fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This grand challenge has mostly proved ineffective because of catalytic mechanism constraints and required chaperone complementarity that hinder Rubisco biogenesis in alternative hosts. Here we refashion Escherichia coli metabolism by expressing a phosphoribulokinase-neomycin phosphotransferase fusion protein to produce a high-fidelity, high-throughput Rubisco-directed evolution (RDE2) screen that negates false-positive selection. Successive evolution rounds using the plant-like Te-Rubisco from the cyanobacterium Thermosynechococcus elongatus BP1 identified two large subunit and six small subunit mutations that improved carboxylation rate, efficiency, and specificity. Structural analysis revealed the amino acids clustered in an unexplored subunit interface of the holoenzyme. To study its effect on plant growth, the Te-Rubisco was transformed into tobacco by chloroplast transformation. As previously seen for Synechocccus PCC6301 Rubisco, the specialized folding and assembly requirements of Te-Rubisco hinder its heterologous expression in leaf chloroplasts. Our findings suggest that the ongoing efforts to improve crop photosynthesis by integrating components of a cyanobacteria CO2-concentrating mechanism will necessitate co-introduction of the ancillary molecular components required for Rubisco biogenesis.

Primeras manifestaciones clínicas de un síndrome de X frágil en la comunidad / First clinical manifestations of a case of fragile X syndrome in the community

Fundamento: el síndrome del X frágil es el más común de los trastornos de retraso mental ligados al cromosoma X. Objetivo: presentar las primeras manifestaciones clínicas de un caso de síndrome de X frágil que no comenzó con retardo del desarrollo psicomotor. Caso clínico: paciente que es remitido a consulta de genética por presentar macrocráneo y orejas displásicas en forma de copa. Al año y seis meses presentó retardo del desarrollo psicomotor. El examen físico, los exámenes complementarios dieron el diagnóstico de un síndrome del X Frágil. Se le puso tratamiento en consulta de estimulación temprana y el paciente mejoró el desarrollo psicomotor. Conclusiones: la aparición de macrocefalia y el retardo del desarrollo psicomotor contribuyeron a realizar el diagnóstico oportuno de esta enfermedad. La estimulación temprana permitió avances en el desarrollo psicomotor del paciente.

Background: fragile X Syndrome is the most common mental retardations disorders linked to X chromosome. Objective: to show the first clinical manifestations of a case of Fragile X Syndrome case that did not began with psychomotor development retardation. Clinical case: a patient who is transferred to genetic consult for presenting macrocranium, dysplastic ears in form of cup. Aged one year and six month old, he had psychomotor development retardation. Physical examinations and complementary test confirmed Fragile X Syndrome diagnosis. The patient was treated in early stimulation consult which improved the psychomotor development retardation. Conclusions: the presence of macrocephaly and later psychomotor development retardation helped to make the appropriate diagnosis of that disorder. Early stimulation permitted advances in psychomotor development in this patient.

Seamless editing of the chloroplast genome in plants.

BACKGROUND: Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. RESULTS: Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein). CONCLUSIONS: Editing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts.

Copper(I) Complexes of Zwitterionic Imidazolium-2-Amidinates, a Promising Class of Electroneutral, Amidinate-Type Ligands.

The first complexes containing imidazolium-2-amidinates as ligands (betaine-type adducts of imidazolium-based carbenes and carbodiimides, NHC-CDI) are reported. Interaction of the sterically hindered betaines ICyCDI(DiPP) and IMeCDI(DiPP) [both bearing 2,6-diisopropylphenyl (DiPP) substituents on the terminal N atoms] with Cu(I) acetate affords mononuclear, electroneutral complexes 1a and 1b, which contain NHC-CDI and acetate ligands terminally bound to linear Cu(I) centers. In contrast, the less encumbered ligand ICyCDI(p-Tol), with p-tolyl substituents on the nitrogen donor atoms, affords a dicationic trigonal paddlewheel complex, [Cu2(µ-ICyCDI(p-Tol))3](2+)[OAc(-)]2 (2-OAc). The nuclear magnetic resonance (NMR) resonances of this compound are broad and indicate that in solution the acetate anion and the betaine ligands compete for binding the Cu atom. Replacing the external acetate with the less coordinating tetraphenylborate anion provides the corresponding derivative 2-BPh4 that, in contrast with 2-OAc, gives rise to sharp and well-defined NMR spectra. The short Cu-Cu distance in the binuclear dication [Cu2(µ-ICyCDI(p-Tol))3](2+) observed in the X-ray structures of 2-BPh4 and 2-OAc, ca. 2.42 Å, points to a relatively strong "cuprophilic" interaction. Attempts to force the bridging coordination mode of IMeCDI(DiPP) displacing the acetate anion with BPh4(-) led to the isolation of the cationic mononuclear derivative [Cu(IMeCDI(DiPP))2](+)[BPh4](-) (3b) that contains two terminally bound betaine ligands. Compound 3b readily decomposes upon being heated, cleanly affording the bis-carbene complex [Cu(IMe)2](+)[BPh4(-)] (4) and releasing the corresponding carbodiimide (C(═N-DiPP)2).

ß-Hydrogen Elimination Reactions of Nickel and Palladium Methoxides Stabilised by PCP Pincer Ligands.

Nickel and palladium methoxides [((iPr)PCP)M-OMe], which contain the (iPr)PCP pincer ligand, decompose upon heating to give products of different kinds. The palladium derivative cleanly gives the dimeric Pd(0) complex [Pd(µ-(iPr)PCHP)]2 ((iPr)PCHP = 2,6-bis(diisopropylphosphinomethyl)phenyl) and formaldehyde. In contrast, decomposition of [((iPr)PCP)Ni-OMe] affords polynuclear carbonyl phosphine complexes. Both decomposition processes are initiated by ß-hydrogen elimination (BHE), but the resulting [((iPr)PCP)M-H] hydrides undergo divergent reaction sequences that ultimately lead to the irreversible breakdown of the pincer units. Whereas the Pd hydride spontaneously experiences reductive C-H coupling, the decay of its Ni analogue is brought about by its reaction with formaldehyde released in the BHE step. Kinetic measurements showed that the BHE reaction is reversible and less favourable for Ni than for Pd for both kinetic and thermodynamic reasons. DFT calculations confirmed the main conclusions of the kinetic studies and provided further insight into the mechanisms of the decomposition reactions.

Excision of plastid marker genes using directly repeated DNA sequences.

Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

Stable plastid transformation of petunia.

Petunia hybrida is a commercial ornamental plant and is also an important model species for genetic analysis and transgenic research. Here we describe the steps required to isolate stable plastid transformants in P. hybrida using the commercial Pink Wave cultivar. Wave cultivars are popular spreading Petunias sold as ground cover and potted plants. Transgenes introduced into P. hybrida plastids exhibit stable expression over many generations. The development of plastid transformation in P. hybrida provides an enabling technology to bring the benefits of plastid engineering, including maternal inheritance and stable expression of performance-enhancing trait genes, to the important floriculture and horticulture industries.