Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacterial CO2-concentrating mechanism, could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

A Paradigm Shift in Primary Liver Cancer Therapy Utilizing Genomics, Molecular Biomarkers, and Artificial Intelligence.

Primary liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related death. Conventional therapies offer limited survival benefit despite improvements in locoregional liver-directed therapies, which highlights the underlying complexity of liver cancers. This review explores the latest research in primary liver cancer therapies, focusing on developments in genomics, molecular biomarkers, and artificial intelligence. Attention is also given to ongoing research and future directions of immunotherapy and locoregional therapies of primary liver cancers.

Chlamydia trachomatis Infection, when Treated during Pregnancy, Is Not Associated with Preterm Birth in an Urban Safety-Net Hospital.

Preterm birth is a major public health problem, occurring in more than half a million births per year in the United States. A number of maternal conditions have been recognized as risk factors for preterm birth, but for the majority of cases, the etiology is not completely understood. Chlamydia trachomatis is one of the most prevalent sexually transmitted infections in the world. However, its role in adverse pregnancy outcome in women is still debated. In order to determine if genitourinary tract infection with C. trachomatis during pregnancy was associated with preterm birth, we conducted a case-control study on women who delivered at Boston Medical Center, an urban "safety-net" hospital that serves a socioeconomically disadvantaged and racially diverse population. Women with known risk factors for preterm birth or immune suppression were excluded. Variables collected on enrolled subjects included demographics; diagnosis of C. trachomatis during or prior to pregnancy; tobacco, alcohol, and illicit substance use; gestational age; and birthweight and gender of the newborn. We also collected urine for chlamydia testing at the time of delivery and placental biopsies for nucleic acid amplification and histological studies. A total of 305 subjects were enrolled: 100 who delivered preterm and 205 who delivered full term. Among those subjects, we identified 19 cases of pregnancy-associated C. trachomatis infection: 6/100 preterm and 13/205 full term, a difference which was not statistically significant. Only two cases of untreated chlamydia infection were identified postpartum, and both occurred in women who delivered at term. We conclude that genitourinary tract infection with C. trachomatis during pregnancy, when appropriately treated, is not associated with preterm birth.

The Chlamydomonas reinhardtii proteins Ccp1 and Ccp2 are required for long-term growth, but are not necessary for efficient photosynthesis, in a low-CO2 environment.

The unicellular green alga Chlamydomonas reinhardtii acclimates to a low-CO2 environment by modifying the expression of a number of messages. Many of the genes that increase in abundance during acclimation to low-CO2 are under the control of the putative transcription factor Cia5. C. reinhardtii mutants null for cia5 do not express several of the known low-CO2 inducible genes and do not grow in a low-CO2 environment. Two of the genes under the control of Cia5, Ccp1 and Ccp2 , encode polypeptides that are localized to the chloroplast envelope and have a high degree of similarity to members of the mitochondrial carrier family of proteins. Since their discovery, Ccp1/2 have been candidates for bicarbonate uptake proteins of the chloroplast envelope membrane. In this report, RNA interference was successful in dramatically decreasing the abundance of the mRNAs for Ccp1 and Ccp2 . The abundance of the Ccp1 and Ccp2 proteins were also reduced in the RNAi strains. The RNAi strains grew slower than WT in a low-CO2 environment, but did not exhibit a mutant carbon concentrating phenotype as determined by the cells' apparent affinity for dissolved inorganic carbon. Possible explanations of this RNAi phenotype are discussed.

Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii.

Carbonic anhydrases (CA) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. The three evolutionarily unrelated families of CAs are designated alpha-, beta-, and gamma-CA. Aquatic photosynthetic organisms have evolved different forms of CO2 concentrating mechanisms (CCMs) to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five CAs have previously been identified in Chlamydomonas reinhardtii, a green alga with a well-studied CCM. Here we identify a sixth gene encoding a beta-type CA. This new beta-CA, designated Cah6, is distinct from the two mitochondrial beta-CAs in C. reinhardtii. Nucleotide sequence data show that the Cah6 cDNA contains an open reading frame encoding a polypeptide of 264 amino acids with a leader sequence likely targeting the protein to the chloroplast stroma. We have fused the Cah6 open reading frame to the coding sequence of maltose-binding protein in a pMal expression vector. The purified recombinant fusion protein is active and was used to partially characterize the Cah6 protein. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate Cah6 from the maltose-binding protein and the purified Cah6 protein was used to raise an antibody. Western blots, immunolocalization studies, and northern blots collectively indicated that Cah6 is constitutively expressed in the stroma of chloroplasts. A possible role for Cah6 in the CCM of C. reinhardtii is proposed.