Studies on the Second Emerson Effect in the Hill Reaction in Algal Cells

This paper shows that the “second Emerson effect”¹ exists not only in photosynthesis, but also in the quinone reduction (Hill reaction), in Chlorella pyrenoidosa and Anacystis nidulans. The peaks at 650 mμ, 600 mμ, 560 mμ, 520 mμ, and 480 mμ, observed in the action spectrum of this effect in the Hill reaction in Chorella, are attributable to chlorophyll b; the occurrence of an additional peak at 670 mμ, 620 mμ, and of two (or three) peaks in the blueviolet region suggests that (at least) one form of chlorophyll a contributes to it. In analogy to suggestions made previously in the interpretation of the Emerson effect in photosynthesis, these results are taken as indicating that excitation by light preferentially absorbed by one (or two) forms of chlorophyll a (Chl a 690 + 700), needs support by simultaneous absorption of light in another form of chlorophyll a (Chl a 670)—directly or via energy transfer from chlorophyll b—in order to produce the Hill reaction with its full quantum yield. In Anacystis, the participation of phycocyanin in the Emerson effect in the Hill reaction is revealed by the occurrence, in the action spectrum of this effect, of peaks at about 560 mμ, 610 mμ, and 640 mμ; a peak at 670 mμ, due to Chl a 670, also is present.     

Action Spectrum for the Appearance of the 520 mμ Difference Band in Illuminated Chlorella Cells

An action spectrum of the 520 mμ difference band in Chlorella is determined using dim illumination. Pigment (or pigments) absorbing most strongly at and above 680 mμ, probably the so-called “long-wave forms” of chlorophyll a appear to be the primary sensitizer of the 520 mμ effect.     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

Photochemical and Nonphotochemical Reactions of Phytochrome in vivo

The nonphotochemical reactions of phytochrome in the coleoptiles of dark-grown corn seedlings were studied at 3 temperatures: 14°, 24°, and 34°. The data obtained show that the destruction of P_fr is the only measurable reaction occurring; reversion of P_fr to P_r was not found. The Q₁₀'s (2.7 and 3.5) and zero order kinetics found for the destruction reaction are consistent with the hypothesis that the reaction is enzyme-mediated.

In vivo action spectra for phytochrome transformation in the coleoptiles of darkgrown corn seedlings were obtained which agree qualitatively with those obtained by other workers for phytochrome-mediated physiological responses and in vitro action spectra. In vivo conversion of phytochrome by blue light, as determined from spectrophotometric measurements of phytochrome itself, is reported. Action peaks for P_r were found at 667 mμ and in the blue in the region of 400 mμ, with a broad shoulder from 590 mμ to 640 mμ. Action peaks for P_fr were found at 725 mμ and in the blue in the region of 400 mμ with a minor peak at 670 mμ, and a broad shoulder from 590 mμ to 640 mμ. The ratio of the quantum efficiencies of P_r at 667 mμ and P_fr at 725 mμ (Φ_r667/Φ_fr725) was estimated to be 1.0.

     

Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen

Aim

Triple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light.

Method

After validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 after injection and 100 J/cm² of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm² on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm² on day 2, which were performed two times every week (“two split” NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2, which were performed three times per week (“three split” NIR-PIT).

Result

Both specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, “two split” NIR-PIT; p < 0.01, “three split” NIR-PIT; p < 0.001) compared with control groups.

Conclusion

NIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

The phylogenetic affiliation and physiological characteristics (e.g., K_s and maximum specific growth rate [μ_max]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.     

Purine Analogue Sensitivity and Lipase Activity of Leptospires

The genus Leptospira can be divided into three groups based on purine analogue sensitivity and lipase (trioleinase) activity. Group 1 contains members of the “parasitic complex” of leptospires which initially cannot grow in media containing 10 μg of 2,6-diaminopurine (DAP) per ml or 200 μg of 8-azaguanine per ml. In addition, leptospires in this group possess lipase activity. Group 2 also contains members of the “parasitic complex” of leptospires. Although these leptospires are similarly sensitive to 8-azaguanine, they differ from group 1 leptospires in that they grow in media containing 10 μg of DAP per ml, and they do not possess detectable lipase activity. Group 3 consists of leptospires belonging to the “biflexa complex.” These leptospires are resistant to both purine analogues and have lipase activity.     

Use of Hydrogenophaga pseudoflava Penetration To Quantitatively Assess the Impact of Filtration Parameters for 0.2-Micrometer-Pore-Size Filters

Filters rated as having a 0.2-μm pore size (0.2-μm-rated filters) are used in laboratory and manufacturing settings for diverse applications of bacterial and particle removal from process fluids, analytical test articles, and gasses. Using Hydrogenophaga pseudoflava, a diminutive bacterium with an unusual geometry (i.e., it is very thin), we evaluated passage through 0.2-μm-rated filters and the impact of filtration process parameters and bacterial challenge density. We show that consistent H. pseudoflava passage occurs through 0.2-μm-rated filters. This is in contrast to an absence of significant passage of nutritionally challenged bacteria that are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry.The 0.2-μm-pore-size filter class (0.2-μm-rated filters) includes a large and diverse set of products (22). They include air filters, particle reduction filters, filters used for bioburden reduction, lab-grade filters, and “sterilizing-grade” filters used in sterile-dosage-form manufacture. ASTM F 838-05, the Brevundimonas diminuta challenge test, is a standard for the “sterilizing-grade” filters (4), a subset of the 0.2-μm-rated filters. The “0.2-μm” designation is applied to the larger and more diverse set of products. This designation is based on physical measurements (e.g., the bubble point, the force necessary to extrude air through the capillary network of a wet filter) and mathematical extrapolations (5, 14, 29).The current filter validation approach for parenteral pharmaceuticals involves a demonstration of removal of 7 log₁₀ CFU/cm² of nutritionally starved B. diminuta from bulk drug product liquids (4, 8, 11, 29). B. diminuta can penetrate 0.2-μm-rated filters, but only sporadically and at low levels (12, 21). Larger bacteria (Listeria monocytogenes) have been demonstrated to be able to penetrate 0.2-μm filters after long-term exposure (27). Recently, a species of small waterborne bacteria, Hydrogenophaga pseudoflava, has been shown to penetrate 0.2-μm-rated filters (31-36) to a greater extent than the above-described bacteria. None of these bacteria are actually physically smaller than 0.2 μm, even H. pseudoflava (25, 37, 38).Because H. pseudoflava penetrates 0.2-μm-rated filters in a potentially quantifiable manner, it can be used to study filtration efficiency. In this report, we evaluate the impact of filtration process parameters and bacterial challenge density on passage. We benchmark H. pseudoflava passage against that of nutritionally challenged bacteria which are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry.     

Measurement of the fluorescence lifetime of chlorophyll a in vivo

New measurements have been made of fluorescence lifetime (τ) of chlorophyll a in the algae Chlorella pyrenoidosa, Porphyridium cruentum, Anacystis nidulans, and in spinach chloroplast. τ-values of 0.6 and 0.7 nsec were obtained with green plants. Anacystis and Porphyridium gave a τ of 0.5 nsec. The previously described two stage decay of fluorescence in vivo in these organisms could not be confirmed. This observation could have been caused by a second wave of light emission from the exciting hydrogen lamp (not detected in earlier work). The lifetimes found in this study (calculated, as before, by the method of convolution integrals) were close to those found by other observers for “low” excitation intensities; the value first reported from this laboratory (1.0-1.7 nsec) may have corresponded to “high” excitation intensity.     

Relationship between the Absorption and Emission Spectra and the "Red Drop" in the Action Spectra of Fluorescence In Vivo

The Stepanov equation, relating the intensity of emission, f_e($\bar{v}$), at a given frequency, and that of absorption, k($\bar{v}$), at the same frequency, is applied, in its modified form (see equation 3 in text) to suspensions of Chlorella, Porphyridium, and Anacystis and to chlorophyll solutions. This application can reveal whether the yield of fluorescence, Φ($\bar{v}$), is constant, or changes with frequency. In Chlorella (green alga) a sharp drop of Φ($\bar{v}$) is indicated towards the lower frequencies (longer waves), beginning around $\bar{v}$ = 1.48 × 10⁴cm^-1 (680 mμ); the Φ($\bar{v}$) function calculated from the Stepanov equation is in fair agreement with the directly determined action spectrum for the excitation of chlorophyll fluorescence in this organism. In Porphyridium (red alga) and Anacystis (blue-green alga) application of the Stepanov equation supports the conclusions, derived from direct measurements, of a much earlier “red drop” of the fluorescence excitation spectra. Direct measurements suggest that the drop in Porphyridium may begin at about 1.53 × 10⁴cm^-1 (654 mμ); in Anacystis, it may begin already above 1.57 × 10⁴cm^-1 (<637mμ). These results confirm the relation, postulated earlier by Duysens and others, between the action spectra of photosynthesis and of chlorophyll a fluorescence in algal cells. The relation of these findings to spectroscopic evidence, suggesting the existence of two main chlorophyll a components in vivo, in green as well as in red and blue-green algae, is discussed.     

Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger Strains Isolated from Grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and β-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 μg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 μg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 μg/liter), and none of the 19 strains of Aspergillus “uniseriate” produced ochratoxin A above the level of detection (4 μg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.     

Thymineless Death and Ultraviolet Sensitivity in Micrococcus radiodurans

Thymine-requiring mutants of Micrococcus radiodurans have been isolated by selection on solid medium containing trimethoprim. Strains requiring either high concentrations of thymine (50 μg/ml) or low concentrations (2 μg/ml) for normal growth were obtained. The Thy⁻ mutant requiring low thymine concentrations has been characterized. It was shown to retain the high ultraviolet light (UV) resistance typical of wild-type M. radiodurans, but it was not resistant to thymineless death. Preliminary exposure of the cells to thymineless conditions resulted in enhanced UV sensitivity, and this interaction occurred under conditions where “unbalanced growth” was inhibited by the addition of chloramphenicol. Upon addition of thymine to deprived cells, UV resistance was gradually restored, and this recovery took place in the absence of protein synthesis. A model is proposed to account for the similarity of thymineless death in bacteria whose deoxyribonucleic acid repair efficiencies differ widely.     

Changes in Chlorophyll a/b Ratio and Products of CO(2) Fixation by Algae Grown in Blue or Red Light

Chlamydomonas and Chlorella were grown for 10 days in white light. 955 μw/cm² blue light (400-500 mμ) or 685 μw/cm² red light (above 600 mμ). Rates of growth in blue or red light were initially slow, but increased over a period of 5 days until normal growth rates were reestablished. During this adaptation period in blue light, total chlorophyll per volume of algae increased 20% while the chlorophyll a/b ratio decreased. In red light no change was observed in the total amount of chlorophyll or in the chlorophyll a/b ratio. After adaptation to growth in blue light and upon exposure to ¹⁴CO₂ with either blue or white light for 3 to 10 minutes, 30 to 36% of the total soluble fixed ¹⁴C accumulated in glycolate-¹⁴C which was the major product. However, with 1 minute experiments, it was shown that phosphate esters of the photosynthetic carbon cycle were labeled before the glycolate. Glycolate accumulation by algae grown in blue light occurred even at low light intensity. After growth of the algae in red light, ¹⁴C accumulated in malate, aspartate, glutamate and alanine, whereas glycolate contained less than 3% of the soluble ¹⁴C fraction.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.     

Can the Red-Green Duochrome Test Be Used Prior to Correcting the Refractive Cylinder Component?

Purpose

A primary task of the eye care professional is determining the refraction, or optical correction, of a patient. The duochrome red-green test is a standard tool for verification of the final refraction. Traditionally, it is recommended for use both prior to and subsequent to determining the cylindrical or astigmatic component of the refraction. In order for it to be effective when used before correcting the cylinder it is necessary that the COLC (Circle of Least Confusion) be on the retina. This study examined whether it is necessarily true that the duochrome response in uncorrected astigmatism will be as trust-worthy as it is with corrected cylinders.

Methods

The red-green examination was performed monocularly under the following three conditions: a. fully corrected refraction for the subgroup of eyes that had spherical refractions and for the subgroup of eyes with sphero-cylindrical refractions. b. best sphere-only correction without cylinder correction in sphero-cylindrical eyes c. an induced cylinder error in spherical eyes. The interval between the last “red” response and the first “green” response for the right eyes as a group and separately for the physiological cylinder and induced cylinder correction sub-groups was calculated and compared using a paired, two-tailed t-test.

Results

The intervals between “red” and “green” responses were not significantly different in the population as a whole and in the uncorrected physiological cylinder and induced cylinder subgroups examined.

Conclusion

Based on the finding that the interval of red-green equality with fully corrected cylinder and without the cylindrical correction are not significantly different, the red-green duochrome test can indeed be used both before and after cylindrical correction.     

Carbon gain and photosynthetic response of chrysanthemum to photosynthetic photon flux density cycles

Most models of carbon gain as a function of photosynthetic irradiance assume an instantaneous response to increases and decreases in irradiance. High- and low-light-grown plants differ, however, in the time required to adjust to increases and decreases in irradiance. In this study the response to a series of increases and decreases in irradiance was observed in Chrysanthemum × morifolium Ramat. “Fiesta” and compared with calculated values assuming an instantaneous response. There were significant differences between high- and low-light-grown plants in their photosynthetic response to four sequential photosynthetic photon flux density (PPFD) cycles consisting of 5-minute exposures to 200 and 400 micromoles per square meter per second (μmol m⁻²s⁻¹). The CO₂ assimilation rate of high-light-grown plants at the cycle peak increased throughout the PPFD sequence, but the rate of increase was similar to the increase in CO₂ assimilation rate observed under continuous high-light conditions. Low-light leaves showed more variability in their response to light cycles with no significant increase in CO₂ assimilation rate at the cycle peak during sequential cycles. Carbon gain and deviations from actual values (percentage carbon gain over- or underestimation) based on assumptions of instantaneous response were compared under continuous and cyclic light conditions. The percentage carbon gain overestimation depended on the PPFD step size and growth light level of the leaf. When leaves were exposed to a large PPFD increase, the carbon gain was overestimated by 16 to 26%. The photosynthetic response to 100 μmol m⁻² s⁻¹ PPFD increases and decreases was rapid, and the small overestimation of the predicted carbon gain, observed during photosynthetic induction, was almost entirely negated by the carbon gain underestimation observed after a decrease. If the PPFD cycle was 200 or 400 μmol m⁻² s⁻¹, high- and low-light leaves showed a carbon gain overestimation of 25% that was not negated by the underestimation observed after a light decrease. When leaves were exposed to sequential PPFD cycles (200-400 μmol m⁻² s⁻¹), carbon gain did not differ from leaves exposed to a single PPFD cycle of identical irradiance integral that had the same step size (200-400-200 μmol m⁻² s⁻¹) or mean irradiance (200-300-200 μmol m⁻² s⁻¹).     

Mitochondrial Genome Analysis of Primary Open Angle Glaucoma Patients

Primary open angle glaucoma (POAG) is a multi-factorial optic disc neuropathy characterized by accelerating damage of the retinal ganglion cells and atrophy of the optic nerve head. The vulnerability of the optic nerve damage leading to POAG has been postulated to result from oxidative stress and mitochondrial dysfunction. In this study, we investigated the possible involvement of the mitochondrial genomic variants in 101 patients and 71 controls by direct sequencing of the entire mitochondrial genome. The number of variable positions in the mtDNA with respect to the revised Cambridge Reference Sequence (rCRS), have been designated “Segregating Sites”. The segregating sites present only in the patients or controls have been designated “Unique Segregating Sites (USS)”. The population mutation rate (θ = 4N_eμ) as estimated by Watterson’s θ (θ_w), considering only the USS, was significantly higher among the patients (p = 9.8×10⁻¹⁵) compared to controls. The difference in θ_w and the number of USS were more pronounced when restricted to the coding region (p<1.31×10⁻²¹ and p = 0.006607, respectively). Further analysis of the region revealed non-synonymous variations were significantly higher in Complex I among the patients (p = 0.0053). Similar trends were retained when USS was considered only within complex I (frequency 0.49 vs 0.31 with p<0.0001 and mutation rate p-value <1.49×10⁻⁴³) and ND5 within its gene cluster (frequency 0.47 vs 0.23 with p<0.0001 and mutation rate p-value <4.42×10⁻⁴⁷). ND5 is involved in the proton pumping mechanism. Incidentally, glaucomatous trabecular meshwork cells have been reported to be more sensitive to inhibition of complex I activity. Thus mutations in ND5, expected to inhibit complex I activity, could lead to generation of oxidative stress and favor glaucomatous condition.     

Development of a Microbial Model for the Combined Effect of Temperature and pH on Spoilage of Ground Meat, and Validation of the Model under Dynamic Temperature Conditions

The changes in microbial flora and sensory characteristics of fresh ground meat (beef and pork) with pH values ranging from 5.34 to 6.13 were monitored at different isothermal storage temperatures (0 to 20°C) under aerobic conditions. At all conditions tested, pseudomonads were the predominant bacteria, followed by Brochothrix thermosphacta, while the other members of the microbial association (e.g., lactic acid bacteria and Enterobacteriaceae) remained at lower levels. The results from microbiological and sensory analysis showed that changes in pseudomonad populations followed closely sensory changes during storage and could be used as a good index for spoilage of aerobically stored ground meat. The kinetic parameters (maximum specific growth rate [μ_max] and the duration of lag phase [λ]) of the spoilage bacteria were modeled by using a modified Arrhenius equation for the combined effect of temperature and pH. Meat pH affected growth of all spoilage bacteria except that of lactic acid bacteria. The “adaptation work,” characterized by the product of μ_max and λ(μ_max × λ) was found to be unaffected by temperature for all tested bacteria but was affected by pH for pseudomonads and B. thermosphacta. For the latter bacteria, a negative linear correlation between ln(μ_max × λ) and meat pH was observed. The developed models were further validated under dynamic temperature conditions using different fluctuating temperatures. Graphical comparison between predicted and observed growth and the examination of the relative errors of predictions showed that the model predicted satisfactorily growth under dynamic conditions. Predicted shelf life based on pseudomonads growth was slightly shorter than shelf life observed by sensory analysis with a mean difference of 13.1%. The present study provides a “ready-to-use,” well-validated model for predicting spoilage of aerobically stored ground meat. The use of the model by the meat industry can lead to effective management systems for the optimization of meat quality.     

Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V_max and K_m for hydrazine are 6.2 ± 0.3 μmol/min · mg and 5.5 ± 0.6 μM, respectively. Hydrazine (25 μM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 μM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of “Candidatus Kuenenia stuttgartiensis” (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.