Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions.

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo .

Meiosis produces haploid gametes that will give rise to the next diploid generation. Chromosome segregation errors occurring at one or both meiotic divisions result in aneuploidy, which can lead to miscarriages or birth defects in humans. During meiosis I, ring-shaped cohesin complexes play important roles to aid in the proper segregation of homologous chromosomes. While REC8 is a specialized meiosis-specific cohesin that functions to hold sister chromatids together, the role of its vertebrate-specific paralog, RAD21L, is poorly understood. Here we tested if Rad21l1, the zebrafish homolog of human and mouse RAD21L, is required for meiotic chromosome dynamics during oogenesis and spermatogenesis. We found that Rad21l1 is an abundant component of meiotic chromosomes where it localizes to both the chromosome axes and the transverse filament of the synaptonemal complex (SC). Knocking out rad21l1 causes nearly the entire mutant population to develop as fertile males, suggesting the mutation triggers a sex reversal from female to male due to a failure in oocyte production. The rad21l1 −/− mutant males display normal fertility at sexual maturity. Sex reversal was partially suppressed in the absence of tp53, suggesting that the rad21l1 −/− mutation causes defects leading to a Tp53 dependent response, specifically in females. The rad21l1 −/− ;tp53 −/− double mutant females produced elevated rates of decomposing eggs and deformed offspring compared to tp53 −/− controls. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of Spo11 does not suppress the sex reversal phenotype. Overall, our data highlight an exceptional sexually dimorphic phenotype caused by knocking out a meiotic-specific cohesin subunit. We propose that Rad21l1 is required for maintaining the integrity of meiotic chromatin architecture during oogenesis.

Author Summary

A prominent symptom of age-linked reproductive decline in women is the increased rate of miscarriage and birth defects due to aneuploidy. Aneuploidy can arise when chromosomes fail to segregate properly during meiosis, the process of creating haploid gametes from a diploid germ cell. Oocyte progression normally arrests prior to anaphase I, after homologous chromosomes have formed crossovers, but before ovulation, which triggers the first round of segregation. This prolonged arrest makes oocytes especially vulnerable to degradation of meiotic chromosome structure and homolog connections over time. Cohesin complexes play a major role in maintaining the meiotic chromosome architecture. Here we assess the role of the vertebrate-specific Rad21l1 cohesin subunit in zebrafish. We find that while males appear mostly unaffected by loss of Rad21l1, oocyte production is massively compromised, leading to sex reversion to males. Sex reversion can be partially prevented in the absence of Tp53, demonstrating that loss or Rad21l1 leads to a Tp53-dependent response in oocytes. Strikingly, double mutant rad21l1 tp53 females produce large numbers of poor quality eggs and malformed offspring. This demonstrates a cohesin-linked vulnerability in female meiosis not present in males and sheds light on a potential mechanism associated with the decline in female reproductive health.

Meiosis is a specialized cellular program required to create haploid gametes from diploid parent cells. Homologous chromosomes pair, synapse, and recombine in a dynamic environment that accommodates gross chromosome reorganization and significant chromosome motion, which are critical for normal chromosome segregation. In Saccharomyces cerevisiae, Ndj1 is a meiotic telomere-associated protein required for physically attaching telomeres to proteins embedded in the nuclear envelope. In this study, we identified additional proteins that act at the nuclear periphery from meiotic cell extracts, including Nup2, a nonessential nucleoporin with a known role in tethering interstitial chromosomal loci to the nuclear pore complex. We found that deleting NUP2 affects meiotic progression and spore viability, and gives increased levels of recombination intermediates and products. We identified a previously uncharacterized 125 aa region of Nup2 that is necessary and sufficient for its meiotic function, thus behaving as a meiotic autonomous region (MAR). Nup2-MAR forms distinct foci on spread meiotic chromosomes, with a subset overlapping with Ndj1 foci. Localization of Nup2-MAR to meiotic chromosomes does not require Ndj1, nor does Ndj1 localization require Nup2, suggesting these proteins function in different pathways, and their interaction is weak or indirect. Instead, several severe synthetic phenotypes are associated with the nup2Δ ndj1Δ double mutant, including delayed turnover of recombination joint molecules, and a failure to undergo nuclear divisions without also arresting the meiotic program. These data suggest Nup2 and Ndj1 support partially overlapping functions that promote two different levels of meiotic chromosome organization necessary to withstand a dynamic stage of the eukaryotic life cycle.

Meiosis is a specialized cellular program required to create haploid gametes from diploid parent cells. Homologous chromosomes pair, synapse, and recombine in a dynamic environment that accommodates gross chromosome reorganization and significant chromosome motion, which are critical for normal chromosome segregation. In Saccharomyces cerevisiae, Ndj1 is a meiotic telomere-associated protein required for physically attaching telomeres to proteins embedded in the nuclear envelope. In this study, we identified additional proteins that act at the nuclear periphery from meiotic cell extracts, including Nup2, a nonessential nucleoporin with a known role in tethering interstitial chromosomal loci to the nuclear pore complex. We found that deleting NUP2 affects meiotic progression and spore viability, and gives increased levels of recombination intermediates and products. We identified a previously uncharacterized 125 aa region of Nup2 that is necessary and sufficient for its meiotic function, thus behaving as a meiotic autonomous region (MAR). Nup2-MAR forms distinct foci on spread meiotic chromosomes, with a subset overlapping with Ndj1 foci. Localization of Nup2-MAR to meiotic chromosomes does not require Ndj1, nor does Ndj1 localization require Nup2, suggesting these proteins function in different pathways, and their interaction is weak or indirect. Instead, several severe synthetic phenotypes are associated with the nup2Δ ndj1Δ double mutant, including delayed turnover of recombination joint molecules, and a failure to undergo nuclear divisions without also arresting the meiotic program. These data suggest Nup2 and Ndj1 support partially overlapping functions that promote two different levels of meiotic chromosome organization necessary to withstand a dynamic stage of the eukaryotic life cycle.

Both [Co^II(qpy)(H₂O)₂]²⁺ and [Fe^II(qpy)(H₂O)₂]²⁺ (with qpy = 2,2':6',2″:6'',2‴-quaterpyridine) are efficient homogeneous electrocatalysts and photoelectrocatalysts for the reduction of CO₂ to CO. The Co catalyst is more efficient in the electrochemical reduction, while the Fe catalyst is an excellent photoelectrocatalyst ( ACS Catal. 2018, 8, 3411-3417). This work uses density functional theory to shed light on the contrasting catalytic pathways. While both catalysts experience primarily ligand-based reductions, the second reduction in the Co catalyst is delocalized onto the metal via a metal-ligand bonding interaction, causing a spin transition and a distorted ligand framework. This orbital interaction explains the experimentally observed mild reduction potential and slow kinetics of the second reduction. The decreased hardness and doubly occupied d_z²-orbital facilitate a σ-bond with the CO₂-π* in an η¹-κC binding mode. CO₂ binding is only possible after two reductions resulting in an EEC mechanism (E = electron transfer, C = chemical reaction), and the second protonation is rate-limiting. In contrast, the Fe catalyst maintains a Lewis acidic metal center throughout the reduction process because the metal orbitals do not strongly mix with the qpy-π* orbitals. This allows binding of the activated CO₂ in an η²-binding mode. This interaction stabilizes the activated CO₂ via a π-type interaction of a Fe-t_2g orbital and the CO₂-π* and a dative bond of the oxygen lone pair. This facilitates CO₂ binding to a singly reduced catalyst resulting in an ECE mechanism. The barrier for CO₂ addition and the second protonation are higher than those for the Co catalyst and rate-limiting.

Objectives

A robust literature documents generational trends in drug use. We examined the implications of changing national drug use patterns on drug injection histories of diverse people who inject drugs (PWID).

Methods

Drug use histories were collected from 776 active PWID in 2011-13. Using descriptive statistics, we examine drug use initiation by year and birth cohort (BC) differences in drug first injected. A multivariate linear regression model of time to injection initiation ([TTII] (year of first injection minus year of first illicit drug use) was developed to explore BC differences.

Results

The first drug injected by BC changed in tandem with national drug use trends with heroin declining from 77% for the pre-1960's BC to 58% for the 1960's BC before increasing to 71% for the 1990's BC. Multivariate linear regression modeling found that shorter TTII was associated with the 1980's/1990's BC (-3.50 years; 95% Confidence Interval [CI]=-0.79, -6.21) as compared to the 1970's BC. Longer TTII was associated with being female (1.65 years; 95% CI=0.40, 2.90), African American (1.69 years; 95% CI=0.43, 2.95), any substance use treatment prior to injection (4.22 years; 95% CI=2.65, 5.79), and prior non-injection use of drug that was first injected (3.29 years; 95% CI=2.19, 4.40).

Conclusion

National drug trends appear to influence injection drug use patterns. The prescription opiate drug era is associated with shorter TTII. Culturally competent, demographically and generationally-targeted prevention strategies to combat transitions to drug injection are needed to prevent or shorten upstream increases in risky drug use practices on a national level.

Background

Heroin use is increasing in the US. Heroin use may predispose users towards injection routes of drug administration as compared to other illicit substances.

Objective

To explore the relationship between heroin use and drug injection, we compared time from first use to first injection (referred to as time to injection onset by drug [TTIOD]) of heroin, methamphetamine/speed, cocaine, and crack cocaine among people who inject drugs (PWID).

Methods

Age of first use and first injection by drug was collected from 776 PWID. Survival analyses were used to determine TTIOD and to examine demographic factors associated with TTIOD. Cox regression analysis was used to determine demographic factors associated with drug-specific injection onset.

Results

The eventual injection onset rate by the drug was 99% for participants who used heroin, 85% for participants who used methamphetamine/speed, 80% for participants who used powder cocaine, and 38% for participants who used crack cocaine. Hazard ratios for injection use within one year of first use by drug were: 1.37 (median survival time [MST] = 0.61 years) for heroin, 0.66 (MST = 1.10 years) for methamphetamine/speed, 0.50 (MST = 2.93 years) for powder cocaine, and 0.12 (MST = 39.59 years) for crack cocaine. Demographic differences in TTIOD were found for each drug. Demographic differences were found for eventual injection by drug for all substances except heroin.

Conclusion

Among PWID, heroin use was associated with a more rapid transition to injection and a higher rate of eventual heroin injection regardless of demographics. More robust, innovative efforts to reduce heroin use and prevent injection initiation are urgently needed.