Stigma towards women requesting abortion and association with health facility staff facilitation and obstruction of abortion care in South Africa.

Background: Abortion stigma has been shown to influence provider attitudes around abortion and may decrease provider willingness to participate in abortion care, or lead some to obstruct care. However, this link remains understudied. Methods: The present study uses baseline data collected through a cluster-randomized controlled trial in 16 public sector health facilities in South Africa in 2020. A total of 279 clinical and non-clinical health facility workers were surveyed. Primary outcome measures included: 1) willingness to facilitate abortion care in eight hypothetical scenarios, 2) facilitation of abortion care in the last 30 days, and 3) obstruction of abortion care in the last 30 days. Logistic regression models were used to assess the association between level of stigma as measured through the Stigmatizing Attitudes, Beliefs and Actions Scale (SABAS) and the primary outcomes. Results: Overall, 50% of respondents in the sample were willing to facilitate abortion care in each of the eight scenarios, with differences in willingness based on the abortion client's age and personal situation in each scenario. Over 90% reported facilitating abortion care in the last 30 days, but 31% also reported having obstructed abortion care in the last 30 days. Stigma was significantly associated with willingness to facilitate abortion care and actual obstruction of abortion care in the last 30 days. Controlling for covariates, odds of willingness to facilitate abortion care in every scenario decreased with every one-point increase in SABAS score (reflecting more stigmatizing attitudes), and odds of obstructing abortion care increased with every one-point increase in SABAS score. Conclusions: Lower abortion stigma on the part of health facility workers was associated with willingness to facilitate abortion access but not actual facilitation of abortion services. Higher abortion stigma was associated with actual obstruction of an abortion service in the last 30 days. Interventions to reduce stigma towards women seeking abortion, and particularly negative stereotyping, among all health facility staff is key to ensuring equitable and non-discriminatory access to abortion. Trial registration: Retrospectively registered on clinicaltrials.gov (ID: NCT04290832) on February 27, 2020. Plain english summary: The link between stigma against women seeking abortion and decisions around whether to provide, abstain, or obstruct abortion care remains understudied. This paper assesses how stigmatizing beliefs and attitudes towards women seeking abortion in South Africa affects willingness to facilitate abortion care and actual facilitation or obstruction of abortion care in practice. A total of 279 clinical and non-clinical health facility workers were surveyed between February and March 2020. Overall, half of respondents in the sample were willing to facilitate abortion care in each of the eight scenarios, with important differences in willingness by scenario. Almost all respondents reported facilitating an abortion procedure in the last 30 days, but one in three also reported having obstructed abortion care in the last 30 days. More stigmatizing attitudes corresponded to decreased willingness to provide abortion care and increased odds of obstructing abortion care. Results show that stigmatizing attitudes, beliefs, and actions toward women who seek abortion shape how clinical and non-clinical staff in South Africa feel about their participation in abortion services and whether they obstruct this care. Facility staff hold great power in determining whose abortions are facilitated and whose are obstructed, resulting in stigma and discrimination being openly perpetuated. Continuous work to reduce stigma towards women seeking abortion among all health workers is key to ensuring equitable and non-discriminatory access to abortion for all.

Reach, experience, and acceptability of an abortion self-care intervention in Bolivia: a mixed-methods evaluation.

In 2018, Ipas Bolivia launched an abortion self-care (ASC) community intervention with the goal of increasing access to supportive, well-informed abortion support provided by community agents (CAs). Between September 2019 and July 2020, Ipas conducted a mixed-methods evaluation to assess the reach, outcomes, and acceptability of the intervention. We used logbook data maintained by CAs to capture demographic characteristics and ASC outcomes of people supported. We also conducted in-depth interviews with 25 women who had received support and 22 CAs who had provided support. 530 people accessed ASC support through the intervention, most of whom were young, single, educated women accessing abortion in the first trimester. Among the 302 people who self-managed their abortions, 99% reported having a successful abortion. No women reported adverse events. All women interviewed expressed satisfaction with the support provided by the CA and, in particular, with the information, lack of judgement, and respect they felt from CAs. CAs spoke highly about their experience and viewed their participation as a way to increase people's ability to exercise their reproductive rights. Obstacles included experiences of stigma, fears of legal repercussions, and difficulties dispelling misconceptions around abortion. Legal restrictions and abortion stigma continue to complicate access to safe abortion, and findings from this evaluation highlight important avenues for the effectiveness and expansion of ASC interventions, including legal support to people who have abortions and those who provide abortion support, building capacity of people as informed buyers, and ensuring that interventions reach rural and other often under-served people.

No effect of a dairy-based, high flavonoid pre-workout beverage on exercise-induced intestinal injury, permeability, and inflammation in recreational cyclists: A randomized controlled crossover trial.

BACKGROUND: Submaximal endurance exercise has been shown to cause elevated gastrointestinal permeability, injury, and inflammation, which may negatively impact athletic performance and recovery. Preclinical and some clinical studies suggest that flavonoids, a class of plant secondary metabolites, may regulate intestinal permeability and reduce chronic low-grade inflammation. Consequently, the purpose of this study was to determine the effects of supplemental flavonoid intake on intestinal health and cycling performance. MATERIALS AND METHODS: A randomized, double-blind, placebo-controlled crossover trial was conducted with 12 cyclists (8 males and 4 females). Subjects consumed a dairy milk-based, high or low flavonoid (490 or 5 mg) pre-workout beverage daily for 15 days. At the end of each intervention, a submaximal cycling trial (45 min, 70% VO2max) was conducted in a controlled laboratory setting (23°C), followed by a 15-minute maximal effort time trial during which total work and distance were determined. Plasma samples were collected pre- and post-exercise (0h, 1h, and 4h post-exercise). The primary outcome was intestinal injury, assessed by within-subject comparison of plasma intestinal fatty acid-binding protein. Prior to study start, this trial was registered at ClinicalTrials.gov (NCT03427879). RESULTS: A significant time effect was observed for intestinal fatty acid binding protein and circulating cytokines (IL-6, IL-10, TNF-α). No differences were observed between the low and high flavonoid treatment for intestinal permeability or injury. The flavonoid treatment tended to increase cycling work output (p = 0.051), though no differences were observed for cadence or total distance. DISCUSSION: Sub-chronic supplementation with blueberry, cocoa, and green tea in a dairy-based pre-workout beverage did not alleviate exercise-induced intestinal injury during submaximal cycling, as compared to the control beverage (dairy-milk based with low flavonoid content).

Telehealth for Addressing Sexual and Reproductive Health and Rights Needs During the COVID-19 Pandemic and Beyond: A Hybrid Telemedicine-Community Accompaniment Model for Abortion and Contraception Services in Pakistan.

The COVID-19 pandemic led overburdened health care systems to deprioritize essential sexual and reproductive healthcare, including abortion and contraception care, while accelerating shifts in healthcare delivery to digital technologies. However, in many countries, including Pakistan, inequalities in access to digital technologies remain, presenting an opportunity for interventions that both increase access to deprioritized sexual and reproductive health and rights (SRHR) services and overcome the digital divide in delivering digital solutions to those in need of SRHR services. In June 2020, Ipas Pakistan partnered with Sehat Kahani (SK), a local health care NGO and telehealth service, and an existing network of Lady Health Workers (LHWs) to launch a novel hybrid telemedicine-community accompaniment pilot. The model linked women via LHWs with mobile devices to online providers for telemedicine consultations for SRH, including abortion services, contraception, and other gynecological consultations. In June 2020, we trained 98 LHWs and 22 telehealth doctors. Between June 2020 and March 2021, a total of 176 women were referred by LHWs for telehealth consultations. Among women who received abortion services, nearly all (90%) reported complete uterine evacuation. No serious adverse events were reported. Overall satisfaction was high; 81% reported being satisfied, and 86% said it is likely they would recommend the telehealth service to others. Data show that the provision of SRHR services via a telehealth-accompaniment model can be successfully implemented in Pakistan. Outcome data show high satisfaction and good clinical outcomes for women accessing care through this model. However, more data are needed to understand the full potential of this model. Barriers to digital health models, such as poor or inconsistent internet access, remain in places like Pakistan, especially in rural settings. This approach has its limitations but should be considered as an option in settings with similarly established community health networks and inequitable access to digital health.

Factors affecting the persistent use of sharp curettage for abortion in public hospitals in Mexico.

OBJECTIVES: Dilation and curettage is an outdated abortion procedure no longer recommended by the World Health Organization. However, use of dilation and curettage remains high in some countries, including Mexico. We aim to understand the factors that contribute to persistent use of dilation and curettage in Mexico. METHODS: We conducted a mixed-methods study in two phases: (1) secondary quantitative data analysis from 40 Ipas-supported public hospitals in Mexico and (2) 28 in-depth interviews in 9 Ipas-affiliated hospitals with doctors, nurses, and hospital administrators. RESULTS: Among our sample, 41% of abortions less than 13 weeks performed in 2019 were treated with dilation and curettage, while this increased to 67% of abortions at or above 13 weeks. Only 18% of induced abortions were performed with dilation and curettage compared to 44% of post-abortion care procedures. The main factor identified as determining use of dilation and curettage in in-depth interviews was availability of abortion supplies, both in terms of cleaning, storage, and maintenance of supplies and in the budgeting and procurement of supplies. Other factors included confidence in the efficacy of other methods, attitudes toward different methods, skill and training, and perceived benefits to patients. CONCLUSION: Ensuring supplies for recommended abortion methods are available is a key lever for any intervention aimed at reducing dilation and curettage use. However, as the doctor performing the abortion decides which method to use, individual factors such as lack of skill and mistrust in other procedures can become a particularly obstinate barrier to recommended method use. Localizing decision-making power in the hands of doctors is problematic in that it places the doctor's preference above that of the person receiving the abortion. It is important to look deeply at the power structures that contribute to doctor-oriented models of abortion care.

Factors Associated with Support for Adolescent Access to Contraception Among Mexican Catholic Parents.

We used a nationally representative survey of 2186 Mexican Catholic parents to assess two outcomes: support for adolescent access to modern contraception and whether adolescents unaccompanied by an adult should have access to contraceptive methods. A majority (85%) of Mexican Catholic parents support adolescent access to modern contraceptive methods, but there was less support (28%) for access to contraception unaccompanied. Further, our results show strong support (92%) for sex education in schools. Parents who believe that good Catholics can use contraception had higher odds of support for adolescent access and unaccompanied access to modern contraception. Mexican Catholic parents support adolescent access to modern contraception, but support for unaccompanied access to contraception is lower. This may reflect an interest in being involved, and not necessarily opposition to contraceptive use. Measures of Catholicism that focus on behaviors may better explain opinions about adolescent access to contraception.

"We don't want problems": reasons for denial of legal abortion based on conscientious objection in Mexico and Bolivia.

BACKGROUND: The misuse of conscientious objection (CO) is a significant barrier to legal abortion access in many countries, especially in Latin America. We examine the reasons for denial of legal abortion services in Mexico and Bolivia and identify ways to mitigate the misuse of CO. METHODS: We conducted 34 in-depth interviews and 12 focus group discussions in two states in Mexico and four departments in Bolivia. Results were coded and categorized using a thematic analysis approach. RESULTS: Denial of abortion services based on CO is widespread in health facilities in Mexico and Bolivia and is primarily employed for reasons other than moral, religious, or ethical considerations. The main reasons for denial of services based on CO is lack of knowledge about abortion-related laws and fear of legal problems in abortion service provision. Conversely, the main reason to provide services is to comply with relevant laws. Denying services under the guise of CO negatively impacts pregnant people and health care teams, including fewer safe abortion options and increased workload and stigma, respectively. Most respondents cited training and education on abortion law as the foremost way to mitigate the negative impacts of the misuse of CO. CONCLUSIONS: For many health personnel, knowing, understanding, and following the law is reason enough to provide abortion services. Individuals who object due to lack of knowledge about laws and fear of legal problems represent a key population that can be sensitized and equipped with the necessary information and resources to provide legal abortion services.

RESUMEN: ANTECEDENTES: El mal uso de la objeción de conciencia (OC) es una barrera importante para el acceso al aborto aún cuando es legal, en muchos países, especialmente en países en América Latina. Examinamos los motivos de la negación de servicios de aborto legal en México y Bolivia e identificamos formas de mitigar el uso indebido de la OC. MéTODOS: Realizamos 34 entrevistas a profundidad y 12 discusiones en grupo focal en dos estados en México y cuatro departamentos en Bolivia. Los resultados fueron codificados y categorizados utilizando un enfoque de análisis temático. RESULTADOS: La negación de servicios de aborto basados ​​en la OC está muy extendida en los establecimientos de salud en México y Bolivia y se emplea principalmente por razones distintas a las consideraciones morales, religiosas o éticas. Las principales razones para la negación de servicios basados ​​en la OC son la falta de conocimiento sobre las leyes relacionadas con el aborto y el temor a problemas legales en la prestación de servicios de aborto. Por el contrario, la razón principal para proporcionar servicios es cumplir con las leyes pertinentes. Negar servicios bajo la apariencia de OC impacta negativamente a las personas embarazadas y a los equipos de atención médica, incluidas menos opciones de aborto seguro y mayor carga de trabajo y estigma, respectivamente. La mayoría de los encuestados mencionaron la capacitación y educación sobre la ley del aborto como la principal forma de mitigar los impactos negativos del uso indebido de la OC. CONCLUSIONES: Para parte del personal de salud, conocer, comprender y cumplir la ley es motivo suficiente para proporcionar servicios de aborto. Las personas que se oponen debido a la falta de conocimiento sobre las leyes y el miedo a los problemas legales representan una población clave que puede ser sensibilizada y equipada con la información y los recursos necesarios para proporcionar servicios de aborto legal.

Regulating Conscientious Objection to Legal Abortion in Argentina: Taking into Consideration Its Uses and Consequences.

Claims of conscientious objection (CO) have expanded in the health care field, particularly in relation to abortion services. In practice, CO is being used in ways beyond those originally imagined by liberalism, creating a number of barriers to abortion access. In Argentina, current CO regulation is lacking and insufficient. This issue was especially evident in the country's 2018 legislative debate on abortion law reform, during which CO took center stage. This paper presents a mixed-method study conducted in Argentina on the uses of CO in health facilities providing legal abortion services, with the goal of proposing specific regulatory language to address CO based not only on legal standards but also on empirical findings regarding CO in everyday reproductive health services. The research includes a review of literature and comparative law, a survey answered by 269 health professionals, and 11 in-depth interviews with stakeholders. The results from our survey and interviews indicate that Argentine health professionals who use CO to deny abortion are motivated by a combination of political, social, and personal factors, including a fear of stigmatization and potential legal issues. Furthermore, we find that the preeminent consequences of CO are delays in abortion services and conflicts within the health care team. The findings of this research allowed us to propose specific regulatory recommendations on CO, including limits and obligations, and suggestions for government and health system leaders.

Misoprostol knowledge and distribution in Mexico City after the change in abortion law: a survey of pharmacy staff.

OBJECTIVE: First-trimester abortion is widely available in Mexico City since legalisation in 2007, but few data exist surrounding pharmacy staff knowledge and sales practices. We describe misoprostol availability, whether a prescription is required, and knowledge of the legal status of abortion and uses for misoprostol among pharmacy staff in Mexico City. METHODS: Data were collected from 174 pharmacies in Mexico City. One employee at each pharmacy was asked about availability, need for prescription, indications for misoprostol, and sociodemographic information. Our primary outcome was availability of misoprostol. We used descriptive and bivariate statistics to compare knowledge and practices by type of pharmacy and staff gender. PATIENTS AND PUBLIC INVOLVEMENT: No patients were involved in this study. RESULTS: Of the 174 pharmacies, 65 were chain and 109 small independent. Misoprostol was available at 61% of sites. Only 49% of independent pharmacies sold misoprostol, compared with 81.5% of chain pharmacies (p<0.05). Knowledge of indications for misoprostol use was similar. The majority (80%) of respondents knew that abortion was legal in Mexico City, and 44% reported requiring a prescription for sale of misoprostol, with no significant difference between male and female staff or by pharmacy type. CONCLUSIONS: Availability, requirement of a prescription, and knowledge of indications for use of misoprostol varies among pharmacies, resulting in differential access to medical abortion. Pharmacies may be a good place to target education for pharmacy staff and women about safe and effective use of misoprostol for abortion.

Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.

Chilled Potatoes Decrease Postprandial Glucose, Insulin, and Glucose-dependent Insulinotropic Peptide Compared to Boiled Potatoes in Females with Elevated Fasting Glucose and Insulin.

Resistant starch (RS) has been shown to improve postprandial glycemia and insulin sensitivity in adults with metabolic syndrome. RS is found naturally in potatoes, where the amount varies based on cooking method and serving temperature. Thirty females with a mean BMI of 32.8 ± 3.7 kg/m2, fasting glucose of 110.5 mg/dL, and insulin of 10.3 µIU/L, completed this randomized, crossover study. A quantity of 250 g of boiled (low RS) and baked then chilled (high RS) russet potatoes were consumed on two separate occasions. Glycemic (glucose and insulin) and incretin response, subjective satiety, and dietary intake were measured. Results showed that the chilled potato elicited significant reductions at 15 and 30 min in glucose (4.8% and 9.2%), insulin (25.8% and 22.6%), and glucose-dependent insulinotropic peptide (GIP) (41.1% and 37.6%), respectively. The area under the curve for insulin and GIP were significantly lower after the chilled potato, but no differences were seen in glucose, glucagon-like peptide-1, and peptide YY, or overall subjective satiety. A higher carbohydrate and glycemic index but lower fat diet was consumed 48-hours following the chilled potato than the boiled potato. This study demonstrates that consuming chilled potatoes higher in RS can positively impact the glycemic response in females with elevated fasting glucose and insulin.

Capturing compassion: A survey of Mexican Catholics assessing abortion support by reason for abortion and degree of Catholicism.

OBJECTIVE: No standard exists to measure religiosity in abortion studies. We test whether religiosity is associated with support for abortion among Mexican Catholics and whether different measures of degree of Catholicism alter the relationship. STUDY DESIGN: We conducted a nationally representative cross-sectional study using survey data from 2669 self-identified Mexican Catholics (response rate 85%). Our three outcomes were 1) overall support for legal abortion and support for abortion under exceptions, grouped as having 2) traditionally high agreement (rape, life) and 3) low agreement (on demand, socioeconomic). We used logistic regression controlling for sociodemographic covariates to test the association of four measures of Catholicism with our outcomes. RESULTS: The majority of Mexican Catholics support abortion in some circumstances. (90% at least one high agreement exception and 40% at least one low agreement exception). The only measure of degree of Catholicism significantly associated with all our outcomes was belief that a person who helps someone who aborts can be a good Catholic. Those who agreed (versus not) had higher odds of support for abortion for both high agreement (aOR 3.6 [95% CI: 1.7-7.9]) and low agreement (aOR 1.9 [95% CI: 1.3-2.7]) exceptions. Respondents who believe a woman who aborts does not need to confess (aOR 2.1 [95% CI: 1.5-2.8]) or needs to confess to God (aOR 1.4 [95% CI: 1.04-1.8]) versus a priest had higher odds of agreement with at least one low agreement exception. CONCLUSION: Opinion toward abortion among Mexican Catholics is diverse. More nuanced measures of Catholicism are valuable in assessing support for abortion, especially exceptions with low support. IMPLICATIONS: The main reasons women need abortion are precisely those with low support among Mexican Catholics. Focusing on nuanced measures of Catholicism can help us identify areas for shared values and frame messaging to meet the diversity of Catholic opinion. Catholicism is not an unsurmountable barrier to liberalization of abortion law.

Access to abortion under the heath exception: a comparative analysis in three countries.

BACKGROUND: Despite Britain, Colombia, and some Mexican states sharing a health exception within their abortion laws, access to abortion under the health exception varies widely. This study examines factors that result in heterogeneous application of similar health exception laws and consequences for access to legal abortion. Our research adds to previous literature by comparing implementation of similar abortion laws across countries to identify strategies for full implementation of the health exception. METHODS: We conducted a cross-country comparative descriptive study synthesizing data from document and literature review, official abortion statistics, and interviews with key informants. We gathered information on the use and interpretation of the health exception in the three countries from peer-reviewed literature, court documents, and grey literature. We next extracted public and private abortion statistics to understand the application of the law in each setting. We used a matrix to synthesize information and identify key factors in the use of the law. We conducted in-depth interviews with doctors and experts familiar with the health exception laws in each country and analyzed the qualitative data based on the previously identified factors. RESULTS: The health exception is used broadly in Britain, somewhat in Colombia, and very rarely in Mexican states. We identified five factors as particularly salient to application of the health exception in each setting: 1) comprehensiveness of the law including explicit mention of mental health, 2) a strong public health sector that funds abortion, 3) knowledge of and attitudes toward the health exception law, including guidelines for physicians in providing abortion, 4) dissemination of information about the health exception law, and 5) a history of court cases that protect women and clarify the health exception law. CONCLUSIONS: The health exception is a valuable tool for expanding access to legal abortion. Differences in the use of the health exception as an indication for legal abortion result in wide access for women in Britain to nearly no access in Mexican states. Our findings highlight the difference between theoretical and real access to legal abortion. The interpretation and application of the health exception law are pivotal to expanding real access to abortion.

Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin.

The antimalarial drug artemisinin is a natural product produced by the plant Artemisia annua. Extracts of A. annua have been used in Chinese herbal medicine for over two millennia. Following the re-discovery of A. annua extract as an effective antimalarial, and the isolation and structural elucidation of artemisinin as the active agent, it was recommended as the first-line treatment for uncomplicated malaria in combination with another effective antimalarial drug (Artemisinin Combination Therapy) by the World Health Organization (WHO) in 2002. Following the WHO recommendation, the availability and price of artemisinin fluctuated greatly, ranging from supply shortfalls in some years to oversupply in others. To alleviate these supply and price issues, a second source of artemisinin was sought, resulting in an effort to produce artemisinic acid, a late-stage chemical precursor of artemisinin, by yeast fermentation, followed by chemical conversion to artemisinin (i.e., semi-synthesis). Engineering to enable production of artemisinic acid in yeast relied on the discovery of A. annua genes encoding artemisinic acid biosynthetic enzymes, and synthetic biology to engineer yeast metabolism. The progress of this effort, which resulted in semi-synthetic artemisinin entering commercial production in 2013, is reviewed with an emphasis on recent publications and opportunities for further development. Aspects of both the biology of artemisinin production in A. annua, and yeast strain engineering are discussed, as are recent developments in the chemical conversion of artemisinic acid to artemisinin.

Access to Contraception in the Context of Zika: Health System Challenges and Responses.

Women in areas of the Americas with endemic Aedes mosquito populations are at risk for exposure to Zika virus, which can cause fetal brain abnormalities and associated congenital microcephaly. Individual health care providers may encounter health system barriers to providing evidence-based care. We focus on Mexico and the state of Texas to highlight the role of health system factors in contraceptive access in the context of Zika and highlight efforts in Puerto Rico as an example of initiatives to improve access to contraception. In Mexico, states with the highest unmet need for contraception are low-lying coastal states. The government recently announced an investment to combat Zika but made no mention of family planning initiatives to assist women in preventing pregnancy. In Texas, the Department of State Health Services has issued recommendations to help women and men avoid mosquito bites; the issue of whether women should plan or avoid pregnancy is not addressed. Puerto Rico has the largest number of confirmed cases of Zika virus in the U.S. states and territories. Recently, the Centers for Disease Control and Prevention Foundation launched the Zika Contraception Access Network, which provides contraceptives at no cost to participating clinics in Puerto Rico. The Zika virus highlights weaknesses in health systems that make it difficult for women to use contraception if they want to delay births. Women across the globe, with or without Zika virus, need access to contraception to prevent unintended pregnancy, and health care providers require functioning health systems that offer support to ensure access is a reality.

Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas.

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.

Biological and genetic factors regulating natural competence in a bacterial plant pathogen.

For naturally competent bacteria, spatially structured growth can provide an environment for enhanced horizontal gene transfer through transformation and recombination. DNA is often present in the extracellular environment, such as in the extracellular matrix of biofilms, and the lysis of a single cell can result in high local DNA concentrations. Xylella fastidiosa is a naturally competent plant pathogen that typically lives in a surface-attached state, yet previous work characterizing the competence of this organism was conducted with planktonic cells in liquid environments. Here, we show that transformation and recombination efficiencies are two to three orders of magnitude higher for cells grown on solid compared with liquid media, with maximum recombination efficiencies of about 10(-3). Cells were highly competent throughout their exponential growth phase, with no significant change in recombination efficiencies until population growth rates began to slow. Mutations in type IV pili, competency-related, and cell-cell signalling genes significantly impacted the ability of X. fastidiosa to acquire and incorporate DNA. Because X. fastidiosa is highly competent when growing in a surface-attached state, as it does within its insect vectors and host plants, recombination of naturally transformed DNA could be a significant route by which horizontal gene transfer occurs in natural environments.

Effects of DNA size on transformation and recombination efficiencies in Xylella fastidiosa.

Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.

Natural competence and recombination in the plant pathogen Xylella fastidiosa.

Homologous recombination is one of many forces contributing to the diversity, adaptation, and emergence of pathogens. For naturally competent bacteria, transformation is one possible route for the acquisition of novel genetic material. This study demonstrates that Xylella fastidiosa, a generalist bacterial plant pathogen responsible for many emerging plant diseases, is naturally competent and able to homologously recombine exogenous DNA into its genome. Several factors that affect transformation and recombination efficiencies, such as nutrient availability, growth stage, and methylation of transforming DNA, were identified. Recombination was observed in at least one out of every 10(6) cells when exogenous plasmid DNA was supplied and one out of every 10(7) cells when different strains were grown together in vitro. Based on previous genomic studies and experimental data presented here, there is mounting evidence that recombination can occur at relatively high rates and could play a large role in shaping the genetic diversity of X. fastidiosa.

Protein folding during cotranslational translocation in the endoplasmic reticulum.

To test how far into the protein-conducting channel of the translocon complex a nascent polypeptide domain must move before it can fold, we analyzed the folding of in vitro translated products of truncated mRNAs encoding the Semliki Forest virus capsid protease domain (Cp) during translocation into microsomes. Cp folded when the C-terminal linker connecting it to the peptidyltransferase center was 64 amino acids or longer. This means that to fold, Cp must exit the translocon channel. With an uncleaved signal sequence, about one out of four of the Cp domains could undergo folding with a C-terminal linker of only 38-66 amino acids. This suggested that the constraint imposed on folding by the translocon complex may be less stringent for signal-anchored membrane proteins.