A Pair of "ACEs".

The emergence of the COVID-19 viral pandemic has generated a renewed interest in pharmacologic agents that target the renin angiotensin system (RAS). Angiotensin-converting enzyme 1 (ACE1) inhibitors decrease the synthesis of angiotensin II (Ang II) from its precursor angiotensin I and inhibit the breakdown of bradykinin, while Ang II receptor blockers antagonize the action of Ang II at the receptor level downstream. The actions of both classes of drugs lead to vasodilation, a blunting of sympathetic drive and a reduction in aldosterone release, all beneficial effects in hypertension and congestive heart failure. ACE2 cleaves the vasoconstrictor Ang II to produce the anti-inflammatory cytoprotective angiotensin 1-7 (Ang 1-7) peptide, which functions through the G protein-coupled receptor MAS to counteract the pathophysiologic effects induced by Ang II via its receptors, including vasoconstriction, inflammation, hypercoagulation, and fibrosis. SARS-CoV-2 enters human cells by binding ACE2 on the cell surface, decreases ACE2 activity, competes for ACE2 receptor-binding sites, and shifts the RAS toward an overexpression of Ang II, accounting for many of the deleterious effects of the virus. Thus, there is great interest in developing recombinant ACE2 as a therapeutic for prevention or treatment of COVID-19. Notably, ACE2 is highly expressed in the oral cavity, and saliva and dorsum of the tongue are major reservoirs of SARS-CoV-2. Cost-effective methods to debulk the virus in the oral cavity may aid in the prevention of viral spread. Here we review the pharmacology of targeted small molecule inhibitors of the RAS and discuss novel approaches to employing ACE2 as a therapeutic for COVID-19.

Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracture healing.

Human insulin-like growth factor-1 (IGF-1) plays important roles in development and regeneration of skeletal muscles and bones but requires daily injections or surgical implantation. Current clinical IGF-1 lacks e-peptide and is glycosylated, reducing functional efficacy. In this study, codon-optimized Pro-IGF-1 with e-peptide (fused to GM1 receptor binding protein CTB or cell penetrating peptide PTD) was expressed in lettuce chloroplasts to facilitate oral delivery. Pro-IGF-1 was expressed at high levels in the absence of the antibiotic resistance gene in lettuce chloroplasts and was maintained in subsequent generations. In lyophilized plant cells, Pro-IGF-1 maintained folding, assembly, stability and functionality up to 31 months, when stored at ambient temperature. CTB-Pro-IGF-1 stimulated proliferation of human oral keratinocytes, gingiva-derived mesenchymal stromal cells and mouse osteoblasts in a dose-dependent manner and promoted osteoblast differentiation through upregulation of ALP, OSX and RUNX2 genes. Mice orally gavaged with the lyophilized plant cells significantly increased IGF-1 levels in sera, skeletal muscles and was stable for several hours. When bioencapsulated CTB-Pro-IGF-1 was gavaged to femoral fractured diabetic mice, bone regeneration was significantly promoted with increase in bone volume, density and area. This novel delivery system should increase affordability and patient compliance, especially for treatment of musculoskeletal diseases.

Dual-Targeting Approach Degrades Biofilm Matrix and Enhances Bacterial Killing.

Biofilm formation is a key virulence factor responsible for a wide range of infectious diseases, including dental caries. Cariogenic biofilms are structured microbial communities embedded in an extracellular matrix that affords bacterial adhesion-cohesion and drug tolerance, making them difficult to treat using conventional antimicrobial monotherapy. Here, we investigated a multitargeted approach combining exopolysaccharide (EPS) matrix-degrading glucanohydrolases with a clinically used essential oils-based antimicrobial to potentiate antibiofilm efficacy. Our data showed that dextranase and mutanase can synergistically break down the EPS glucan matrix in preformed cariogenic biofilms, markedly enhancing bacterial killing by the antimicrobial agent (3-log increase versus antimicrobial alone). Further analyses revealed that an EPS-degrading/antimicrobial (EDA) approach disassembles the matrix scaffold, exposing the bacterial cells for efficient killing while concurrently causing cellular dispersion and "physical collapse" of the bacterial clusters. Unexpectedly, we found that the EDA approach can also selectively target the EPS-producing cariogenic bacteria Streptococcus mutans with higher killing specificity (versus other species) within mixed biofilms, disrupting their accumulation and promoting dominance of commensal bacteria. Together, these results demonstrate a dual-targeting approach that can enhance antibiofilm efficacy and precision by dismantling the EPS matrix and its protective microenvironment, amplifying the killing of pathogenic bacteria within.

Acid suppressing therapy as a risk factor for Candida esophagitis.

Studies were reviewed from PubMed for risk factors for the development, recurrence, prevention and therapy of Candida esophagitis, and for mechanisms induced by acid-suppressing therapy potentially influencing these factors. Documented observations included greatly increased Candida populations in the mouth, esophagus, stomach, and upper small intestine induced by acid-suppressing therapy. Among patients without HIV disease, PPI consumers more frequently had developed Candida esophagitis than did non-consumers and had also developed its recurrences more frequently. Similar phenomena associated with H2 -blocker use were less intense, and the possibility of similar phenomena in patients with HIV disease apparently had not yet been examined in spite of their high frequency of this disorder. PPI-induced elimination of the gastric acid barrier is a major mechanism leading to oro-pharyngeal and esophageal candida colonization, while PPI-induced impairment of absorption of most orally administered antifungal agents may limit the prophylactic and therapeutic success of these agents. These observations suggest potential value in limiting PPI use in populations of patients with Candida infections including esophagitis, as well as in patients at risk for their development, and also suggest that post-PPI rebound acid hypersecretion may provide additional anti-Candida benefit. Studies designed to develop the risk-benefit ratios of PPI use in these patients deserve investigation with high priority appropriate for studies in patients with HIV disease.

Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality.

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%-31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bio-reactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes.

Although bamboo is one of the most important woody crops in Asia, information on its genome is still very limited. To investigate the relationship among Poaceae members and to understand the mechanism of albino mutant generation in vitro, the complete chloroplast genome of two economically important bamboo species, Dendrocalamus latiflorus Munro and Bambusa oldhamii Munro, was determined employing a strategy that involved polymerase chain reaction (PCR) amplification using 443 novel primers designed to amplify the chloroplast genome of these two species. The lengths of the B. oldhamii and D. latiflorus chloroplast genomes are 139,350 and 139,365 bp, respectively. The organization structure and the gene order of these two bamboos are identical to other members of Poaceae. Highly conserved chloroplast genomes of Poaceae facilitated sequencing by the PCR method. Phylogenetic analysis using both chloroplast genomes confirmed the results obtained from studies on chromosome number and reproductive organ morphology. There are 23 gaps, insertions/deletions > 100 bp, in the chloroplast genomes of 10 genera of Poaceae compared in this study. The phylogenetic distribution of these gaps corresponds to their taxonomic placement. The sequences of these two chloroplast genomes provide useful information for studying bamboo evolution, ecology and biotechnology.

Novel pathways for glycoprotein import into chloroplasts.

Although the chloroplast contains its own genome, majority of its protein components are encoded by nuclear genes and must be imported post-translationally. In general, proteins synthesized by cytosolic ribosomes are post-translationally targeted to the chloroplast through interactions between their N-terminal transit sequence and protein translocon Toc/Tic complexes in the chloroplast membranes. An alternative pathway that mediates post-translational delivery of proteins to the chloroplast via the secretory pathway was recently described. This pathway provides new opportunities for complementation of the chloroplast protein maturation machinery with chaperones needing endoplasmic reticulum and/or Golgi typical maturations such as N-glycosylation for their biological activity or using chloroplasts as a storage compartment for glycoproteins.

Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi.

The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Contrary to previous predictions, accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid composition of plastid membranes compared with the membranes of susceptible target microbes. In vitro assays with protein extracts from T(1) and T(2) plants confirmed that MSI-99 was expressed at high levels to provide 88% (T(1)) and 96% (T(2)) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selection, leaf extracts from T(2) generation plants showed 96% inhibition of growth against P. syringae pv tabaci. In addition, leaf extracts from transgenic plants (T(1)) inhibited the growth of pregerminated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, whereas transformed leaves showed no signs of necrosis, demonstrating high-dose release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showed necrotic anthracnose lesions in non-transformed control leaves, whereas transformed leaves showed no lesions. Genetically engineering crop plants for disease resistance via the chloroplast genome instead of the nuclear genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes.

Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts.

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.

Osteoporosis due to androgen deprivation therapy in men with prostate cancer.

OBJECTIVES: The frequency of osteoporotic fractures is greatly increased in men receiving androgen deprivation therapy (ADT), but whether the risk of osteoporosis differs between different types of ADT or between continuous and intermittent therapy has not been determined. Techniques for modifying ADT-associated bone loss have not been clearly identified. METHODS: Risk factors for the development of osteoporosis in men receiving ADT will be reviewed. Relations between bone mineral density (BMD) values and the development of osteoporotic fractures, along with methods for preventing both BMD loss and osteoporotic fractures, will be discussed. RESULTS: ADT rapidly accelerates bone loss among men with prostate cancer and multiplies the risk of osteoporotic fractures among them. Factors other than ADT-associated bone loss contributing to this fracture risk include both decreased BMD before ADT and an increased tendency to fall associated with muscle weakness, impaired balance, and postural hypotension. Each of these factors may be associated with poor nutrition, advancing malignant disease, hypogonadism of non-ADT origin, advanced age, and the use of narcotic, antihypertensive, or sedative medications. Although the success of therapy designed to improve BMD values and lower the fracture rate in these patients has not been explored, regular exercise, smoking abstinence, adequate calcium, protein, and vitamin D intake, maintenance of weight, and the use of bisphosphonates or calcitonin may each have a useful therapeutic role. Theoretical considerations suggest that intermittent ADT may decrease the frequency of ADT-associated osteoporosis. CONCLUSIONS: An urgent need exists for the definition of techniques useful in preventing osteoporotic fractures in men receiving ADT for prostate cancer.

Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection.

Chloroplast genetic engineering offers several advantages over nuclear transformation including high levels of gene expression and gene containment. However, a consequence of placing a transgene in the chloroplast genome is that the antibiotic resistance genes used as selectable markers are highly amplified. Engineering genetically modified (GM) crops without the use of antibiotic resistance genes should eliminate the potential risk of their transfer to the environment or gut microbes. Therefore, the betaine aldehyde dehydrogenase (BADH) gene from spinach was used in this study as a selectable marker. The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast BADH enzyme to non-toxic glycine betaine, which also serves as an osmoprotectant. Chloroplast transformation efficiency was 25-fold higher in BA selection than with spectinomycin. In addition, rapid regeneration was obtained. Transgenic shoots appeared within 12 days in 80% of leaf disks (up to 23 shoots per disk) under BA selection compared to 45 days in 15% of disks (1 or 2 shoots per disk) under spectinomycin selection. Southern blots confirmed stable integration of foreign genes into all of the chloroplast genomes (approximately 10,000 copies per cell) resulting in homoplasmy. Transgenic tobacco plants showed 15- to 18-fold higher BADH activity at different developmental stages than untransformed controls. Transgenic plants were morphologically indistinguishable from untransformed plants and the introduced trait was inherited stably in the subsequent generation. This is the first report of genetic engineering of the higher plant chloroplast genome without the use of antibiotic selection. The use of naturally occurring genes in spinach for selection, in addition to gene containment, should ease public concerns regarding GM crops.

Hypogonadism following prostate-bed radiation therapy for prostate carcinoma.

BACKGROUND: The degree of testicular damage resulting from primary treatment of prostate carcinoma by external beam radiation therapy (EBRT) to the prostate bed has not been determined. If significant testicular damage has occurred, the resulting endocrine changes may result in modified tumor behavior, contribute to postradiation impotence, and may aggravate other signs and symptoms of hypogonadism, potentially influencing a patient's choice of primary treatment for his tumor. METHOD: Three to eight years after primary treatment for localized prostate carcinoma, serologic evaluation for hypogonadism was undertaken in 33 men who had received EBRT and in 55 similar men who had received radical prostatectomy (RP). No subjects had developed recognized tumor recurrence, and none had undergone hormonal treatment since primary therapy. RESULTS: Among men of similar age, prior treatment with EBRT was associated with significantly more frequent hypogonadism than prior treatment with RP. In men with EBRT, total testosterone levels averaged 27.3% less, free testosterone levels 31.6% less, dihydrotestosterone levels 33.4% less, luteinizing hormone (LH) levels 52.7% greater, and follicle-stimulating hormone (FSH) levels 100% greater than those values in men who had prior treatment with RP. Differences between postradiation and postsurgical men in LH and FSH levels were most prominent in men older than 70 years. CONCLUSIONS: Three to eight years after primary treatment for prostate carcinoma, striking hormone differences were present between men who had received EBRT to the prostate bed and those with prior RP. These differences strongly suggested that prominent and permanent testicular damage was sustained during EBRT, frequently severe enough to cause hypogonadism.

Antibiotic-free chloroplast genetic engineering - an environmentally friendly approach.

Chloroplast genetic engineering offers several advantages over nuclear genetic engineering, including gene containment and hyperexpression. However, introducing thousands of copies of transgenes into the chloroplast genome amplifies the antibiotic resistance genes. Two recent articles report different and novel strategies to either remove antibiotic resistance genes or select chloroplast transformants without using these genes. This should eliminate their potential transfer to microorganisms or plants and ease public concerns about genetically modified crops.

Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants.

The use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. Here, we discuss recent developments in this field and possible environmental concerns.

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals.

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T(0) and T(1) transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.

Progressive osteoporosis during androgen deprivation therapy for prostate cancer.

PURPOSE: Hypogonadism is a prominent risk factor for osteoporosis in older men. However, bone loss during androgen ablation therapy for prostate cancer has rarely been quantitated. MATERIALS AND METHODS: Femoral neck bone mineral density was determined in 26 men before orchiectomy or chemical castration as initial hormone therapy for prostate cancer and at 6-month intervals thereafter for 6 to 42 months. Measurements were made in 16 other men at 12 to 24 months beginning 3 to 8 years after the onset of castration. Baseline and post-castration bone loss was related to several host and tumor characteristics, and compared to similar measurements in 12 control subjects. RESULTS: Average age corrected baseline femoral neck bone mineral density was higher in controls than in treated men and remained essentially unchanged for 2 years. Following orchiectomy average bone mineral density decreased 2.4% and 7.6%, respectively, during years 1 and 2 (2-year loss 2.5% to 17.0%), with similar losses documented in men undergoing chemical castration. Average bone mineral density decreased 1.4% to 2.6% per year 3 to 8 years after uninterrupted androgen deprivation. Age corrected baseline bone mineral density was greater in men who were obese, younger than 75 years or participated in regular exercise but the influence of each characteristic could not be isolated. Post-castration bone loss was greater in men who were obese, younger than 75 years without regular exercise. CONCLUSIONS: Chemical or surgical castration in men with prostate cancer is usually followed by greatly accelerated bone loss which may be superimposed on a bone mass already depleted before hormonal therapy. Baseline bone mass and subsequent bone loss may be influenced by host obesity, age and exercise habits.

Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts.

Bioelastic protein-based polymers (PBP) have several medical (prevention of post-surgical adhesions) and non-medical (biodegradable plastic) applications. This study compares expression levels of PBP genes (synthetic) integrated into the nuclear genome or the large single-copy (LSC) or inverted repeat (IR) region of the chloroplast genome in transgenic tobacco plants. Polymer transcripts accumulated up to 100-fold higher in the IR plants than in those of nuclear transgenic plants. Integration of foreign genes into all of the chloroplast genomes (homoplasmy) and higher levels of polymer transcripts were observed only in the IR and not in LSC transgenic plants. Expression of the polymer protein was further confirmed by Western blot analysis.