Making Security Viral: Shifting Engineering Biology Culture and Publishing.

The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes.

Identification of the tirandamycin biosynthetic gene cluster from Streptomyces sp. 307-9.

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl-tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307-9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase-nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly-line oxidative modifications.

Biofuel alternatives to ethanol: pumping the microbial well.

Engineered microorganisms are currently used for the production of food products, pharmaceuticals, ethanol fuel and more. Even so, the enormous potential of this technology has yet to be fully exploited. The need for sustainable sources of transportation fuels has generated a tremendous interest in technologies that enable biofuel production. Decades of work have produced a considerable knowledge-base for the physiology and pathway engineering of microbes, making microbial engineering an ideal strategy for producing biofuel. Although ethanol currently dominates the biofuel market, some of its inherent physical properties make it a less than ideal product. To highlight additional options, we review advances in microbial engineering for the production of other potential fuel molecules, using a variety of biosynthetic pathways.

Utilizing the power of microbial genetics to bridge the gap between the promise and the application of marine natural products.

Marine organisms are a rich source of secondary metabolites. They have yielded thousands of compounds with a broad range of biomedical applications. Thus far, samples required for preclinical and clinical studies have been obtained by collection from the wild, by mariculture, and by total chemical synthesis. However, for a number of complex marine metabolites, none of these options is feasible for either economic or environmental reasons. In order to proceed with the development of many of these promising therapeutic compounds, a reliable and renewable source must be found. Over the last twenty years, the study of microbial secondary metabolites has greatly advanced our understanding of how nature utilizes simple starting materials to yield complex small molecules. Much of this work has focused on polyketides and nonribosomal peptides, two classes of molecules that are prevalent in marine micro- and macroorganisms. The lessons learned from the study of terrestrial metabolite biosynthesis are now being applied to the marine world. As techniques for cloning and heterologous expression of biosynthetic pathways continue to improve, they may provide our greatest hope for bridging the gap between the promise and application of many marine natural products.

Clinical and histopathological evaluation of 13 cases of adenocarcinoma in aged rhesus macaques (Macaca mulatta).

In recent years, the emphasis on aging research, has led to an increase in the number of aged macaques being maintained in some research facilities with a subsequent increase in the occurrence of age-related diseases. One of the most commonly reported age related diseases is intestinal adenocarcinoma. At the University of Illinois at Chicago (UIC), which maintains a colony of approximately 55 aged rhesus macaques 13 cases of intestinal adenocarcinoma were diagnosed within a 25-month period. This report provides a comprehensive description of the clinical findings for intestinal adenocarcinoma in aged rhesus macaques, including results from physical examinations, laboratory tests, radiographic evaluations, gross and histopathologic findings as well as a comparison with the disease condition in humans. The use of carcinoembryonic antigen as a potential tumor marker was evaluated by immunohistochemical analysis of tissue specimens in 10 cases. Intestinal adenocarcinoma is a disease condition that should be of concern to individuals responsible for the care of aged rhesus macaques.

Novel use of the Elizabethan collar as a "hoop-skirt" for protecting wounds and catheters in nonhuman primates.

This article describes the novel use of an Elizabethan collar, which is attached to a primate jacket to create a tamper-proof "hoop-skirt" for protecting wounds and catheters. We successfully have used this hoop-skirt to manage juvenile male rhesus monkeys with Foley catheters for 10 days post-prostatectomy. In addition, our hoop-skirt has been used to manage wounds on the hindlimbs of both macaques and baboons.

Knowledge of ethical lapses and other experiences on clinical licensure examinations.

While clinical licensure examinations claim to protect the public from incompetent practitioners, serious problems related to issues of validity, reliability, and ethics have been reported. The ethical lapses are difficult to document, and reports of problems have been strictly anecdotal. This study's primary purpose was to verify those anecdotal reports by mailing a twenty-one-item survey to a national random sample of one thousand general dentists who graduated from a United States-accredited dental school between 1980 and 1994. For those who responded (42.9 percent) results show that 23.9 percent did not arrange for follow-up care for the patient even though it was indicated. Eight percent reported knowledge of instances where a lesion was intentionally created; 19.3 percent knew of premature treatment provided for purposes of the examination; 13.7 percent knew of a colleague who coerced a patient into accepting treatment; 32.5 percent reported knowledge of unnecessary radiographs. About half of all respondents agreed that the examination was not an accurate assessment of ability, and a similar proportion doubted that human patients were necessary. A more reliable and valid assessment strategy that does not jeopardize patient care is needed.

Ex vivo expansion of autologous bone marrow CD34(+) cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons.

Hematopoietic stem cell (HSC) self-renewal in vitro has been reported to result in a diminished proliferative capacity or acquisition of a homing defect that might compromise marrow repopulation. Our group has demonstrated that human HSC expanded ex vivo in the presence of porcine microvascular endothelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further test the marrow repopulating capacity of expanded stem cells, our laboratory has established a myeloablative, fractionated total body irradiation conditioning protocol for autologous marrow transplantation in baboons. A control animal, which received no transplant, as well as two animals, which received a suboptimal number of marrow mononuclear cells, died 37, 43, and 59 days postirradiation, respectively. Immunomagnetically selected CD34(+) marrow cells from two baboons were placed in PMVEC coculture with exogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granulocyte-macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobblestone area-forming cells (CAFC) over input. Both baboons became transfusion independent by day 23 posttransplant and achieved absolute neutrophil count (ANC) >500/microL by day 25 +/- 1 and platelets >20,000/microL by day 29 +/- 2. This hematopoietic recovery was delayed in comparison to two animals that received either a graft consisting of freshly isolated, unexpanded CD34(+) cells or 175 x 10(6)/kg unfractionated marrow mononuclear cells. Analysis of the proliferative status of cells in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of CD34(+) cells present in the cultures had undergone cycling, and that the population of cells expressing a CD34(+) CD38(-) phenotype in the cultures was also the result of active cell division. These data indicate that isolated bone marrow CD34(+) cells may undergo cell division during ex vivo expansion in the presence of endothelial cells to provide a graft capable of rescuing a myeloablated autologous host.

Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity.

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for alpha-smooth muscle actin (alphaSMA). Expression of alphaSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of alphaSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.

Extracoelomic fluid osmometry and electrolyte composition during early gestation in the baboon.

OBJECTIVE: Access to extracoelomic fluid offers the opportunity to assess and potentially treat genetic disorders early in pregnancy. We have been using the pregnant baboon as a model to develop techniques and evaluate the feasibility of sampling extracoelomic fluid. The aim of this study was to determine the osmolality, oncotic pressures, and electrolyte composition of the baboon's extracoelomic fluid between days 39 and 41 of gestation and to compare them with those of maternal blood. STUDY DESIGN: The optimal time to perform the coelocentesis procedure was determined in 14 timed pregnant baboons. Six coelocenteses were then performed in aseptic conditions, under continuous transvaginal ultrasonographic guidance and avoiding the amniotic or yolk sacs. Between 3 and 5 mL extracoelomic fluid was aspirated from each baboon with a 10-mL syringe. Only 1 attempt at sampling was performed for each of the 6 animals. Pregnancies were tracked by transabdominal ultrasonographic evaluations on postprocedure day 3 and then weekly until day 140 of pregnancy. Oncotic pressures and biochemical measurements were determined with 1 mL extracoelomic fluid and 1 mL heparinized maternal venous blood. RESULTS: Data analysis suggests that maternal blood and extracoelomic fluid have similar osmolalities and concentrations of electrolytes but different colloid osmotic pressures (P <.001). CONCLUSION: This nonhuman primate model can be used to gain some insight into the physiologic changes in the composition of the extracoelomic fluid and to evaluate the safety of the coelocentesis procedure. The data suggest that the chorion laeve behaves as a semipermeable membrane at 40 days' gestation.

Assessment of the risk of fetal loss after the coelocentesis procedure using a baboon model.

OBJECTIVES: The aims of this study were to gain some insight with regard to the safety of the coelocentesis procedure and, to determine pH, pCO2, and base excess status of the extracoelomic fluid 40 days after fertilization. METHODS: Twenty-eight timed-mated baboons from the breeding colony of the Biological Resource Laboratory at the University of Illinois at Chicago were studied. The initial 19 animals were used to determine the ultrasonographic relations between the different compartments of the gestational sac from 38 to 62 days of pregnancy. Under aseptic conditions, in 9 animals coelocenteses were then performed, under continuous transvaginal ultrasound guidance to avoid the amniotic or yolk sacs. Extracoelomic fluid (1-5 ml) was aspirated using 10-ml syringes. Only one attempt of sampling was performed in each of the 9 animals. Extracoelomic fluid pH, pCO2, and base excess were compared with maternal femoral venous blood. Pregnancies were followed by transabdominal ultrasound evaluations on day 3 after the procedure and weekly until day 140 after fertilization. RESULTS: Extracoelomic fluid could be aspirated easily using a 20-gauge needle. Only one pregnancy loss was detected within 3 days after the procedure. No complications occurred in the remaining 8 pregnancies. Extracoelomic fluid pH (7.45 +/- 0.01) and pCO2 (39.9 +/- 2.4 mm Hg) were significantly different from maternal femoral venous blood pH (7.41 +/- 0.01) and pCO2 (47.1 +/- 1.4 mm Hg; p < 0.02), while base excess values were similar. CONCLUSIONS: This preliminary study performed at 40 days after fertilization in the baboon model suggests that the coelocentesis procedure is technically simple and presents a relatively low risk to mother and fetus if a 20-gauge needle is used and the amount of aspirated extracoelomic fluid is <3 cm3. At this gestational age, the extracoelomic fluid is more alkalotic than maternal femoral venous blood.

Newborn primate infants are entrained by low intensity lighting.

At the present time we do not know when the circadian timing system of human infants becomes responsive to light. Because of human study limitations, it is not currently possible to address this issue in clinical studies. Therefore, to provide insights into when the circadian system of humans becomes responsive to light, baboons were studied. We first assessed if the biological clock located in suprachiasmatic nuclei (SCN) is responsive to light at birth. When term newborn infants were exposed to bright light at night (5000 lux), SCN metabolic activity and c-fos mRNA expression increased, indicating the presence of photic responsiveness. When photic entrainment of developing rhythmicity was examined in infants, low intensity (200 lux) cycled lighting was sufficient to entrain circadian phase. However, low intensity lighting was not sufficient to induce changes in SCN metabolic activity or c-fos mRNA expression. Phase-response studies indicated that light exposure (200 lux) before the onset of activity most effectively shifted circadian phase. These data provide direct evidence that the SCN are responsive to visually mediated light information in a primate at birth. Further consideration of lighting conditions that infants are exposed to is therefore warranted.

Transvaginal ultrasonographic (TVS) evaluation of baboon gestation from 37-62 days postconception.

Our objective was to determine the growth of the embryo and surrounding structures during baboon (Papio anubis) gestation using transvaginal sonography (TVS). To this end, we evaluated 19 timed-mated baboons using TVS between 37 and 62 days of gestation. After visualization of the gestational sac, amniotic sac, and yolk sac, the three largest diameters of each of these extra embryonic structures were measured using longitudinal and transverse views. Embryonic crown-rump length (CRL) was also recorded. Embryonic heart rates were determined using the M-mode function of the ultrasound equipment. All 19 gestations developed without complications. No significant trend could be demonstrated for heart rate or yolk sac diameters over the 37-62 day gestational age period. Mean (SD) gestational age in days, heart rate, and yolk sac diameter, respectively, for the group were 48 (7.8) days (range: 37-61), 180 (15) beats per minute (range: 156-221) and 5 (0.1) mm (range: 3-8). Significant correlations (P < 0.0001) were determined between gestational age and CRL and gestational and amniotic sacs. We conclude that TVS allows a clear visualization of the embryo proper and all the cavities within the gestational sac of the baboon gestation. This study has determined the normal pattern of changes of these cavities from 37-62 days of gestation. Future applications of these findings may include sampling fluid from these cavities for biochemical, cytological, and metabolic studies.

Comparative microanatomy of the lateral wall of the 'cavernous sinus' in humans and the olive baboon.

Despite many studies of the 'cavernous sinus' lateral wall, the anatomy of this area remains controversial. We performed a comparative microanatomical and histoarchitectural study in 14 humans and in 10 nonhuman primates (Papio cynocephalus anubis). Venous channels and cranial nerves were embedded in the 'interperiosteodural space'. The dura propria of the lateral wall could be removed without entering the venous compartment. The oculomotor and trochlear nerves were accompanied by an arachnoidal and dural sheath. The oculomotor nerve sheath stopped under the anterior clinoid process in baboons. The trigeminal ganglion was covered posteriorly with an arachnoid membrane and adhered firmly to the dura propria on lateral and anterior sections. The three branches of the trigeminal nerve had no arachnoid covering, except for arachnoid granulations in humans. In baboons, the oculomotor and trochlear nerves were thicker than in humans, while the ophthalmic nerve was thinner. The abducens nerve belonged to the lateral wall of the sinus in baboons and had no arachnoidal sheath except in the first millimeters of Dorello's canal. After leaving their arachnoidal and dural sheath, the intracavernous cranial nerves acquired a typical peripheral sheath. The venous channels in both species were true dural sinuses. Willis cords and adipose tissue were identified.

Chorionic gonadotropin, estradiol, and progesterone levels in baboons (Papio anubis) during early pregnancy and spontaneous abortion.

The purpose of this study was to sequentially characterize peripheral chorionic gonadotropin (CG), estradiol (E2), and progesterone (P) profiles during early pregnancy in the baboon (Papio anubis). Ten pregnant baboons were bled sequentially at eighteen time points between Days 8 and 128 of gestation. In addition, blood was obtained at corresponding time points from 5 spontaneously aborting baboons. CG levels were assessed in a mouse Leydig cell bioassay using rhesus pituitary LH as the standard E2 and P levels were measured by RIA. Pregnancy-associated CG activity was detectable by Day 15 (901.4 +/- 275.6 ng/ml), peaked at Day 27 (53,494.1 +/- 14,995.6 ng/ml), and then returned to baseline values by Day 51 (45.4 +/- 8.9 ng/ml). Mean E2 concentrations rose from 28.9 +/- 4.3 pg/ml on Day 8 to 280.6 +/- 145.5 pg/ml on Day 58 and then increased 9-fold to a level of 2436.4 +/- 928.0 pg/ml on Day 72. Mean E2 concentrations ranged between 2065.9 and 3830.9 pg/ml from Day 72 through Day 128. Mean P concentrations ranged from 7.5 to 10.2 ng/ml between Days 8 and 21, rising dramatically to 38.6 +/- 4.6 ng/ml on Day 37 prior to declining to levels that ranged from 11.1 to 17.1 ng/ml between Days 51 and 128. CG levels were low in 3 of 5 animals prior to spontaneous abortion E2 and P concentrations preceding spontaneous abortion were similar to values in uncomplicated pregnancy; however, at the time abortion was detected P concentrations in all 5 animals were 1.3 ng/ml or less.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma corticotropin-releasing factor concentrations in the baboon during pregnancy.

We have studied the secretion of placental CRF during pregnancy in the baboon, an animal model with many similarities to human pregnancy. Plasma CRF was measured in two groups of animals. In group 1, studies were performed in six anesthetized animals beginning 8 days postconception. In group 2, studies were performed in five unanesthetized chronically catheterized maternal and five fetal animals in the latter third of pregnancy. In the first study beginning early in pregnancy, CRF was undetectable in all animals on days 8 and 15 postconception. Plasma CRF became detectable in two animals on day 24 and in the remaining four on day 30. Plasma CRF rose significantly to a mean of 810 +/- 160 pg/ml at 37 days gestation (F = 4.20; P < 0.001). Mean maternal plasma CRF was 2452 +/- 1120 pg/ml on day 44 and remained elevated, with a great deal of variability between subjects, until the end of the study period (128 days of gestation). Samples in this group were obtained after ketamine sedation. The effect of ketamine on CRF was studied in three chronically catheterized animals. Samples were obtained before and 2, 4, 6, and 24 h after ketamine administration (40 mg, iv). The baseline CRF concentration was 1168 +/- 131 pg/ml and did not change significantly over the time period studied. In the second study in the chronically catheterized animals, maternal plasma CRF was 1990 +/- 680 pg/ml at 131-140 days gestation and remained elevated until near term at 170 days (term = 175-180 days). Within 24 h after birth, plasma CRF became undetectable (< 60 pg/ml). CRF was also measured in chronically catheterized fetal baboons. The mean CRF concentration was 614 +/- 224 pg/ml at 131-140 days and remained in this range until the end of the period studied (151-160 days gestation). To characterize the CRF immunoactivity in maternal baboon plasma, Sephadex chromatography was performed on an 8.4-ml plasma sample obtained at 160 days gestation. The majority of the CRF immunoactivity eluted in the same position as synthetic human CRF. We conclude that high levels of placental CRF are present in the systemic circulation of the maternal and fetal baboon during pregnancy. In contrast to human pregnancy, which is characterized by an exponential rise in maternal CRF concentrations in the final weeks before delivery, an exponential rise in maternal baboon CRF concentrations occurs early in pregnancy.