Robust and stable transcriptional repression in Giardia using CRISPRi.

Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

A kinematic and kinetic analysis of drop landings in military boots.

OBJECTIVE: The purpose of this study was to examine knee valgus in drop landings during three different footwear conditions and to examine the ground reaction forces exhibited during the drop landing in the three different footwear conditions. METHODS: Sixteen male and female Reserve Officer Training Corps (ROTC) university undergraduate cadets (21 +/- 3 yrs, 79 +/- 12 kg, and 172 +/- 10 cm) volunteered to participate in the study. Kinematic data were collected while participants performed drop landings in three conditions: bare feet, tennis shoes, and issued military boots. RESULTS: Significant differences among footwear for ground reaction forces (bare feet: 1646 +/- 359%, tennis shoe: 1880 +/- 379%, boot: 1833 +/- 438%; p < 0.05) were found, while there was no significant difference in knee valgus among footwear. CONCLUSIONS: Though footwear conditions did not affect knee valgus, they did affect ground reaction forces. Participants in this study had yet to receive any military training on how to land properly from a specified height. Further research should be completed to analyze the kinematics and kinetics of the lower extremity during different landing strategies implemented by trained military personnel in order to better understand injury mechanisms of drop landings in this population. It is likely that injury prevention landing techniques would be beneficial if these were employed by the military and not just in the sporting community.

Diagnosis of pulmonary embolus using ventilation/perfusion lung scintigraphy: more than 0.5 segment of ventilation/perfusion mismatch is sufficient.

BACKGROUND: To determine the optimal diagnostic cut-off point using a simplified criterion for the detection of pulmonary embolus (PE) and to evaluate the criterion's utility and reporter reproducibility. METHODS: Lung scintigraphy was carried out in 924 patients for the diagnosis of PE. This group consisted of 316 men and 608 women with median age of 63 years (range 18-94 years). Ventilation imaging was carried out with Tc-99m Technegas followed by perfusion imaging using 190 MBq Tc-99m macroaggregated albumin. Studies were classified using a 6-category probability criterion of incremental ventilation/perfusion (V/Q) mismatch: A, normal; B, low (minor matched V/Q defects or segmental matched V/Q defects without opacity on chest X-ray); C, low-moderate (a partial segment of V/Q mismatch); D, moderate (1 segment of mismatch); E, moderate-high (1-2 segments of V/Q mismatch) and F, high probability (=2 segments of V/Q mismatch). Clinical end-points at 3 and 6 months were death by PE or PE treated with anticoagulation therapy. Three-reporter reproducibility was determined by kappa statistic on a subgroup of patients (53/924). RESULTS: A total of 122 patients (13%) had a confirmed diagnosis of PE at 3 months and no additional cases were registered at 6 months. The lung scintigraphy probability classification showed: normal 152 (16%), low 620 (67%), low-moderate 20 (2%), moderate 28 (3%), moderate-high 24 (3%) and high 80 (9%). The respective sensitivities and specificities, where the diagnostic cut-offs were established at F, high; E, moderate-high; D, moderate and C, low-moderate probability, were F, 64 and 100%; E, 82 and 99%; D, 95 and 98% and C, 98 and 96%. The respective false-negative cases for F, E, D and C cut-offs were 44, 22, 7 and 3. Using the revised Prospective Investigation of Pulmonary Embolism Diagnosis reporting classification reporter agreement showed kappa values of 0.31-0.48. Using a simplified 2-category (>0.5 segment of V/Q mismatch positive, all others negative) criterion resulted in a higher reporting agreement (kappa 0.74-0.83). There were only 3% of indeterminate cases if this was defined by the D category and a maximum of 8% if categories C, D and E were included. CONCLUSIONS: Using a simplified diagnostic criterion where all studies showing >0.5 segments of V/Q mismatch are regarded as positive and all others as negative, lung scintigraphy, incorporating Tc-99m Technegas ventilation imaging or its equivalent, can achieve a very high diagnostic accuracy for the detection of PE. Using this technique, less than 5% of scans are indeterminate. A simplified, unambiguous approach to reporting is recommended.

Complementary role of adjunctive breast magnetic resonance imaging and scintimammography in patients of all ages undergoing breast cancer surgery.

The aims of this study were to assess breast MRI and scintimammography (SMM) for the detection of breast cancer, and to determine any complementary role of these tests to each other and conventional imaging. Seventy-two patients (age 35-81 years) with a suspicious breast mass were investigated by mammography, breast ultrasound, breast MRI and SMM before undergoing surgical excision of the breast mass. Sensitivity, specificity and area under receiver operator characteristic curves were calculated for each test. Of the 72 patients, there were 66 proven malignant tumours, including two patients with bilateral breast cancer. When comparing the diagnostic sensitivity of breast MRI and SMM for lesion size, both tests showed higher sensitivities for lesions >25 mm in size, particularly for SMM. When these tests were compared for patient age, patients less than 51 years showed higher sensitivities for both tests. This was statistically significant for breast MRI. The overall respective diagnostic sensitivities for mammography, mammography with breast ultrasound, breast MRI and SMM were 56, 67, 86 and 85%. The differences were significant between mammography, mammography/ultrasound and both breast MRI and SMM. Breast MRI and SMM offer incremental diagnostic advantage in the diagnosis of breast cancer. Although improved diagnostic accuracy is seen in patients of all ages, those patients less than 51 years of age receive the greatest diagnostic benefit.

The Horizontal Gaze Nystagmus test: fraudulent science in the American courts.

The Horizontal Gaze Nystagmus (HGN) test was conceived, developed and promulgated as a simple procedure for the determination of the blood alcohol concentration of drivers suspected of driving while intoxicated (DWI). Bypassing the usual scientific review process and touted through the good offices of the federal agency responsible for traffic safety, it was rushed into use as a law enforcement procedure, and was soon adopted and protected from scientific criticism by courts throughout the United States. In fact, research findings, training manuals and other relevant documents were often held as secrets by the state. Still, the protective certification of its practitioners and the immunity afforded by judicial notice failed to silence all the critics of this deeply flawed procedure. Responding to criticism, the sponsors of the test traveled the path documented in this paper that led from mere (if that word can ever truly apply to a matter of such gravity) carelessness in research through self-serving puffery and finally into deliberate fraud--always at the expense of the citizen accused.

Determination of the optimal minimum radioiodine dose in patients with Graves' disease: a clinical outcome study.

The study was performed under the auspices of the International Atomic Energy Commission, Vienna, Austria, with the aim of determining the optimal minimum therapeutic dose of iodine-131 for Graves' disease. The study was designed as a single-blinded randomised prospective outcome trial. Fifty-eight patients were enrolled, consisting of 50 females and 8 males aged from 17 to 75 years. Each patient was investigated by clinical assessment, biochemical and immunological assessment, thyroid ultrasound, technetium-99m thyroid scintigraphy and 24-h thyroid 131I uptake. Patients were then randomised into two treatment groups, one receiving 60 Gy and the other receiving 90 Gy thyroid tissue absorbed dose of radioiodine. The end-point markers were clinical and biochemical response to treatment. The median follow-up period was 37.5 months (range, 24-48 months). Among the 57 patients who completed final follow-up, a euthyroid state was achieved in 26 patients (46%), 27 patients (47%) were rendered hypothyroid and four patients (7%) remained hyperthyroid. Thirty-four patients (60%) remained hyperthyroid at 6 months after the initial radioiodine dose (median dose 126 MBq), and a total of 21 patients required additional radioiodine therapy (median total dose 640 MBq; range 370-1,485 MBq). At 6-month follow-up, of the 29 patients who received a thyroid tissue dose of 90 Gy, 17 (59%) remained hyperthyroid. By comparison, of the 28 patients who received a thyroid tissue dose of 60 Gy, 17 (61%) remained hyperthyroid. No significant difference in treatment response was found (P=0.881). At 6 months, five patients in the 90-Gy group were hypothyroid, compared to two patients in the 60-Gy group (P=0.246). Overall at 6 months, non-responders to low-dose therapy had a significantly larger thyroid gland mass (respective means: 35.9 ml vs 21.9 ml) and significantly higher levels of serum thyroglobulin (respective means: 597.6 microg/l vs 96.9 microg/l). Where low-dose radioiodine treatment of Graves' disease is considered, a dose of 60 Gy will yield a 39% response rate at 6 months while minimising early hypothyroidism. No significant advantage in response rate is gained by using a dose of 90 Gy. For more rapid therapeutic effect at the expense of an increased rate of hypothyroidism, doses in excess of 120 Gy may be required. Ultrasound determination of thyroid mass and measurement of serum thyroglobulin levels may be predictive of those patients who will be less responsive to low-dose therapy.

End-position nystagmus as an indicator of ethanol intoxication.

The Horizontal Gaze Nystagmus test is used by law enforcement agencies in the United States to determine whether drivers are intoxicated. It has a high baseline error and a dose/response relationship that varies greatly according to whether the subject's blood alcohol concentration is rising or falling. Confusion exists among practitioners of the test about whether it quantifies alcohol concentration or evaluates impairment. Fatigue exacerbates one component of the HGN test, end-position nystagmus. Video tapes recorded by cameras in police vehicles revealed that police officers rarely comply with the minimum requirements of the nystagmus examination procedures for which they were trained and certified.

Detection of widespread fluids in the Tibetan crust by magnetotelluric studies.

Magnetotelluric exploration has shown that the middle and lower crust is anomalously conductive across most of the north-to-south width of the Tibetan plateau. The integrated conductivity (conductance) of the Tibetan crust ranges from 3000 to greater than 20,000 siemens. In contrast, stable continental regions typically exhibit conductances from 20 to 1000 siemens, averaging 100 siemens. Such pervasively high conductance suggests that partial melt and/or aqueous fluids are widespread within the Tibetan crust. In southern Tibet, the high-conductivity layer is at a depth of 15 to 20 kilometers and is probably due to partial melt and aqueous fluids in the crust. In northern Tibet, the conductive layer is at 30 to 40 kilometers and is due to partial melting. Zones of fluid may represent weaker areas that could accommodate deformation and lower crustal flow.

The effects of donepezil on the P300 auditory and visual cognitive evoked potentials of patients with Alzheimer's disease.

Donepezil is a cholinesterase inhibitor used for the treatment of patients with mild to moderately severe Alzheimer's disease (AD). The purpose of this study was to determine the effect of treatment with donepezil 5 mg qd on cognitive evoked potentials (EPs) of patients with AD. Although treatment with donepezil did not normalize EP latencies, treatment was associated with a significant decrease in the auditory P300 latency (mean latency pretreatment=401. 5 msec; posttreatment=392.7 msec.; P=0.04), and the visual P300 latency (mean latency pretreatment=605.7 msec; posttreatment=580.3 msec; P=0.04). Treatment with donepezil had no discernible effect on auditory or visual P300 EP amplitudes.

The InterCon network: a program for education partnerships at the University of Texas-Houston Health Science Center.

The University of Texas-Houston Health Science Center (UT-Houston) has created programs and activities to address the state's pressing needs in minority education. Through InterCon, a network of universities and K-12 schools, UT-Houston works with its partners to identify competitive candidates in the current pool of minority graduates with bachelor's degrees and to help them--along with their non-minority counterparts--progress in their education. Another objective is to expand the pool of minorities underrepresented in medicine who complete high school and go to college. In 1994 UT-Houston and Prairie View A&M University created a collaborative venture to provide new educational opportunities at UT-Houston for Prairie View's predominantly African American students. A three-track summer internship program--a result of that collaboration--has since been expanded to partnerships with other minority and majority universities throughout Texas. In 1998, for example, 108 undergraduate students from these universities (and 40 other universities nationwide) participated in research, professional, and administrative summer internships at UT-Houston. The InterCon network also has partnerships with K-12 schools. UT-Houston works with inner-city, suburban, and rural school districts to develop education models that can be transferred throughout the state. The partnerships deal with helping to teach basic academic skills and computer literacy, improve science-related instruction, meet demands for health promotion materials and information for school-initiated health and wellness programs, and develop distance-learning paradigms. UT-Houston views InterCon as a program helping Texas institutions to engage and adapt to the socioeconomic factors, demographic changes, and technology explosion that currently challenge public education.

Expression and function of Fas on cells damaged by gamma-irradiation in B6 and B6/lpr mice.

Fas (CD95) is a cell surface protein that mediates apoptosis. lpr is a mutation of the Fas gene caused by a retroviral insertion resulting in premature termination of transcription and aberrant splicing of Fas mRNA. Mice homozygous for the lpr gene develop lymphoproliferation and produce autoantibodies closely resembling those of human systemic lupus erythematosus. While lpr mice have been reported to express low levels of normally spliced Fas mRNA, it is unknown whether they express functional Fas protein. Here we show that splenocytes from lpr mice that have been damaged by gamma-irradiation expressed Fas protein. Fas was up-regulated on irradiated B6 cells and could be detected on B6/lpr cells undergoing apoptosis following in vitro culture. Detection of Fas on live lpr cells was demonstrable when apoptosis was blocked by zinc. In a short term chimera system, Fas was shown to play a role, in vivo, in the disposition of radiation-injured cells from both normal and lpr mice. The addition of anti-Fas Ab to in vitro cultures resulted in an increase in apoptosis in both B6 and B6/lpr cells. Detection of intact Fas message and low levels of Fas protein in lpr mice has led to the consideration of lpr as a leaky mutation. This study demonstrates that lpr mice can produce functional Fas protein. This system is also appropriate for identifying the in vivo role of Fas/FasL in apoptosis following other cell manipulations.

Methodological issues in evaluating a culture-based substance abuse treatment program in the Kuskokwim Delta, Alaska.

This paper presents a rationale for using videotape records in evaluation research. Although videotaping is commonly used in ethnographic and sociometric analysis, the evaluation literature does not show the application of these recording techniques to program evaluations. The authors outline a model for exploring the effectiveness of video in qualitative program evaluation.

Radiation and stress-induced apoptosis: a role for Fas/Fas ligand interactions.

The lpr gene encodes a defective form of Fas, a cell surface protein that mediates apoptosis. This defect blocks apoptotic deletion of autoreactive T and B cells, leading to lymphoproliferation and lupus-like autoantibody production. The effects of the lpr Fas mutation on other kinds of physiologically relevant apoptosis are largely undocumented. To assess whether some of the apoptosis known to occur after ionizing radiation might be mediated by Fas/Fas ligand (FasL) interactions, we quantitated in vitro apoptosis by flow cytometry measurement of DNA content in splenic T and B cells from irradiated 5- to 8-month-old B6/lpr mice. Total apoptosis of both lpr and control cells was substantial after treatment; however there was a significant difference between B6 (73%) and lpr (25%) lymphocyte apoptosis. Thy1, CD4, CD8, and IgM cells from lpr showed much lower levels of apoptosis than control cells after irradiation. Apoptosis induced by heat shock was also impaired in lpr. The finding that gamma-irradiation increased Fas expression on B6 cells and that irradiation-induced apoptosis could be blocked with a Fas-Fc fusion protein further supported the possible involvement of Fas in this form of apoptosis. Fas/FasL interactions may thus play an important role in identifying and eliminating damaged cells after gamma-irradiation and other forms of injury.

Partially Molten Middle Crust Beneath Southern Tibet: Synthesis of Project INDEPTH Results

INDEPTH geophysical and geological observations imply that a partially molten midcrustal layer exists beneath southern Tibet. This partially molten layer has been produced by crustal thickening and behaves as a fluid on the time scale of Himalayan deformation. It is confined on the south by the structurally imbricated Indian crust underlying the Tethyan and High Himalaya and is underlain, apparently, by a stiff Indian mantle lid. The results suggest that during Neogene time the underthrusting Indian crust has acted as a plunger, displacing the molten middle crust to the north while at the same time contributing to this layer by melting and ductile flow. Viewed broadly, the Neogene evolution of the Himalaya is essentially a record of the southward extrusion of the partially molten middle crust underlying southern Tibet.

Surface geometry of retrieved McKee-Farrar total hip replacements.

A coordinate measurement machine was used to determine the surface geometry of 22 retrieved McKee-Farrar total hip replacements. The radial clearance of each acetabular cup-femoral head pair was computed to see whether there was potential for hydrodynamic lubrication. The surface geometry was used to estimate the volumetric wear for the acetabular cup and the femoral head, and to assess whether those prostheses that had the potential for fluid film lubrication also displayed lower wear. The volumetric wear rate for each prosthesis was estimated by dividing the volumetric wear by the corresponding service life. Two distinct wear patterns were seen on the femoral head. Prostheses displaying polar wear on the femoral head showed significantly greater service lives than those displaying non-polar wear. Although several prostheses had clearances that provided for the possibility of hydrodynamic lubrication, no correlation could be found between clearance and the measured wear.

Semantic and visual factors affecting the representation of map information.

In past research evidence has been found for both mental imagery and propositional hierarchies in subjects' map representations. How the visual and semantic factors associated with maps influence the use of one form of representation or another was the subject of the study reported here. Subjects were required to make relational judgments about city pairs. The results of experiment 1 indicated that superordinate relationships (the relationship of the counties of which the cities were members) affected only the most difficult perceptual judgments, but affected all judgments made from memory. Experiment 2 was done to determine the extent to which these findings were attributable to perceptual distortions rather than propositionally stored rules. Results of experiment 3 showed the degree of perceptual distinction necessary for propositional rules to have no significant influence on relational judgments.

A seasonal pattern of hospital medication errors in Alaska.

Specific behavioral consequences of seasonal affective disorder have not been closely examined. Length of daylight is evaluated in relation to medication errors in a medical center located in the far north. Factors such as numbers of patient admissions, discharges, and deaths were controlled with data collected in Anchorage, Alaska, over 5 consecutive years, 1985-89. These data revealed that 58% of all medication errors occurred during the first quarter of the year. Medication errors were 1.95 times more likely in December than September. The best statistical prediction was for errors associated with levels of darkness 2 months earlier. There may be not only an impairment of work performance among hospital nursing staff that reaches a peak in late winter but, more importantly, medication errors appear to follow a pattern that is closely associated with the annual cycle of daylight and darkness.