Manuscript

Manuscript. Figure ___. Membrane transport characteristics of Xenopus laevis oocytes transformed with clone#3992. mRNA encoding orf3992 was transcribed in vitro from plasmid pEP(clone#3992) and transformed into Xenopus laevis oocytes. (A) Sugar transport into oocytes in the presence and absence of 10 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Extracellular medium supplemented with 1 mM sugar was left with oocytes for 1 hour and then intracellular concentration of sugar was measured. (B) Measurement of beemolulose transport kinetics into oocytes in the presence of different sugars.

 

 

 

Figure ____. Modifications to orf3992 within A. candicus. (A) Schematic showing CRISPR/Cas9 with guide RNA (gRNA) that targets orf3992 co-transformed into A. candicus. (B) Effect of CRISPR/Cas9 disruption of orf3992 gene on A. candicus growth characteristics. Sugars were added to minimal growth medium and A. candicus was grown for 12 hours in minimal medium plus sugar. (C) Schematic showing transformation of expression plasmid encoding orf3992 into A. candicus. (D) Effect of orf3992 overexpression on A. candicus growth in minimal medium plus sugars.

 

Figure ____. Screening Austral candicus cDNA library. (A) cDNA library preparation diagram. mRNA was extracted from A. candicus, converted to cDNA via reverse-transcription, followed by cloning into an expression plasmid with promoter constitutively active within S. cerevisiae. Following transformation of plasmid library into S. cerevisiae, 109 clones were plated onto minimal medium with all amino acids added lacking any sugar but beemolulose. Resulting colonies were picked and grown in 96-well liquid format with the same medium as used for plating colonies. Clone #3992 was one of 20 colonies that showed growth in the 96-well plate, had the most robust growth, and was chosen for further study. (B) Growth of yeast strains on different sugars. Growth was in liquid minimal medium supplemented with sugar(s) indicated. In the right panel, equimolar combinations of beemolulose plus indicated sugar were supplemented into minimal growth medium.

 

 

Figure ____. Identification and cellular localisation of ORF3992 protein. (A) Sequencing of clone #3992 reveals an open reading frame (ORF3992) of 700 amino acids long with hydropathy plotting potentially showing transmembrane domains. (B) Adding FLAG-tag to C-terminus of ORF3992 followed by transformation into yeast strain W303. (C) SDS-PAGE analysis of cytoplasm, membrane, and nucleus fractions from (-) yeast cells alone and (+) yeast cells carrying pEP(clone #3992). (D) Western blot of cytoplasm, membrane, and nucleus fractions from (-) yeast cells alone and (+) yeast cells carrying pEP(clone #3992). Probed with a rabbit anti-FLAG tag primary antibody, followed by anti-rabbit secondary antibody, and visualised with enhanced chemiluminescence (ECL) reagent.

 

 

 

 

 

 

 

B. Sugar

0

1750

3500 Tr

an sp

or t R

at e

(p m

ol /m

in /o

oc yt

e)

A ra

bi no

se

X yl

os e

B ee

m ol

ul os

e

La ct

os e

F ru

ct os

e

R ha

m no

se

M an

no se

S uc

ro se

G lu

co se

CCCP (10 µM)

Control A.

Substrate 1 Substrate 2 Km (mM) Vmax (pmols/min/oocyte)

Beemolulose – 20 ± 1 3499 ± 380 Beemolulose Glucose 22 ± 2 3298 ± 350 Beemolulose Mannose 21 ± 1 3507 ± 373 Beemolulose Rhamnose 102 ± 5 3445 ± 401

Beemolulose Transport Characteristics

 

B.

Sugar

0

1750

3500

T

r

a

n

s

p

o

r

t

 

R

a

t

e

(

p

m

o

l

/

m

i

n

/

o

o

c

y

t

e

)

A

r

a

b

i

n

o

s

e

X

y

l

o

s

e

B

e

e

m

o

l

u

l

o

s

e

L

a

c

t

o

s

e

F

r

u

c

t

o

s

e

R

h

a

m

n

o

s

e

M

a

n

n

o

s

e

S

u

c

r

o

s

e

G

l

u

c

o

s

e

CCCP (10 µM)

Control

A.

Substrate 1Substrate 2Km (mM)Vmax (pmols/min/oocyte)

Beemolulose- 20 ± 1 3499 ± 380

BeemoluloseGlucose22 ± 2 3298 ± 350

BeemoluloseMannose 21 ± 13507 ± 373

BeemoluloseRhamnose102 ± 5 3445 ± 401

Beemolulose Transport Characteristics

 

A.

La ct

os e

R ha

m no

se

B ee

m ol

ul os

e

X yl

os e

M an

no se

F ru

ct os

e

S uc

ro se

G lu

co se

N on

e

Sugar added to minimal medium

0

Δorf3992 Wild-type

100

150

G ro

w th

(% )

C.

B.

pEP(clone #3992)

La ct

os e

R ha

m no

se

B ee

m ol

ul os

e

X yl

os e

M an

no se

F ru

ct os

e

S uc

ro se

G lu

co se

N on

e

Sugar added to minimal medium

0

pEP(clone#3992) Wild-type

100

200

G ro

w th

(% )

Cas9+ gRNA

orf3992

D.

 

A.

L

a

c

t

o

s

e

R

h

a

m

n

o

s

e

B

e

e

m

o

l

u

l

o

s

e

X

y

l

o

s

e

M

a

n

n

o

s

e

F

r

u

c

t

o

s

e

S

u

c

r

o

s

e

G

l

u

c

o

s

e

N

o

n

e

Sugar added to minimal medium

0

Δorf3992

Wild-type

100

150

G

r

o

w

t

h

 

(

%

)

C.

B.

pEP(clone #3992)

L

a

c

t

o

s

e

R

h

a

m

n

o

s

e

B

e

e

m

o

l

u

l

o

s

e

X

y

l

o

s

e

M

a

n

n

o

s

e

F

r

u

c

t

o

s

e

S

u

c

r

o

s

e

G

l

u

c

o

s

e

N

o

n

e

Sugar added to minimal medium

0

pEP(clone#3992)

Wild-type

100

200

G

r

o

w

t

h

 

(

%

)

Cas9+

gRNA

orf3992

D.

 

A ra

bi no

se

X yl

os e

La ct

os e

R ha

m no

se

A ra

bi no

se

X yl

os e

+ Beemolulose

B ee

m ol

ul os

e

La ct

os e

F ru

ct os

e

R ha

m no

se

M an

no se

M an

no se

S uc

ro se

S uc

ro se

G lu

co se

G lu

co se

109 independent clones

Minimal medium + beemolulose

B.

A.

Sugar(s) added to minimal medium

0

Yeast + pEP(clone #3992)

Yeast

50

100

G ro

w th

(% )

mRNA

Saccharomyces cerevisae

Austral candicus

Expression Plasmid (pEP)

cDNA

Yeast + pEP(clone #3992)

Yeast

 

A

r

a

b

i

n

o

s

e

X

y

l

o

s

e

L

a

c

t

o

s

e

R

h

a

m

n

o

s

e

A

r

a

b

i

n

o

s

e

X

y

l

o

s

e

+ Beemolulose

B

e

e

m

o

l

u

l

o

s

e

L

a

c

t

o

s

e

F

r

u

c

t

o

s

e

R

h

a

m

n

o

s

e

M

a

n

n

o

s

e

M

a

n

n

o

s

e

S

u

c

r

o

s

e

S

u

c

r

o

s

e

G

l

u

c

o

s

e

G

l

u

c

o

s

e

10

9

independent clones

Minimal medium

+ beemolulose

B.

A.

Sugar(s) added to minimal medium

0

Yeast + pEP(clone #3992)

Yeast

50

100

G

r

o

w

t

h

 

(

%

)

mRNA

Saccharomyces

cerevisae

Austral candicus

Expression

Plasmid

(pEP)

cDNA

Yeast + pEP(clone #3992)

Yeast

 

C.

220

KDa

100 120

80

50

60

40

20

30

MW M

ar ke

r

Cytoplasm Membrane Nucleus – + – + – + D.

220

KDa

100 120

80

50

60

40

20

30

MW M

ar ke

r

Cytoplasm Membrane Nucleus – + – + – +

Western Blot 1° antibody: anti-FLAG

SDS-PAGE

pEP(clone #3992)

B.

pEP(clone #3992)

FLAG-tag

W303(pEP(clone #3992))

A.

ORF3992 Position of Protein

H yd

ro pa

th y

+3

0

+2 +1

-1 -2 -3

1 508254

N- -C

 

C.

220

KDa

100

120

80

50

60

40

20

30

M

W

 

M

a

r

k

e

r

CytoplasmMembraneNucleus

+

+

+

D.

220

KDa

100

120

80

50

60

40

20

30

M

W

 

M

a

r

k

e

r

CytoplasmMembraneNucleus

+

+

+

Western Blot

1° antibody: anti-FLAG

SDS-PAGE

pEP(clone #3992)

B.

pEP(clone #3992)

FLAG-tag

W303(pEP(clone #3992))

A.

ORF3992

Position of Protein

H

y

d

r

o

p

a

t

h

y

+3

0

+2

+1

-1

-2

-3

1

508

254

N- -C

Manuscript

 
"Looking for a Similar Assignment? Get Expert Help at an Amazing Discount!"

Biology(Hormone)

Biology(Hormone).

  1. A patient having renal surgery suddenly develops massive hypertension and dies during surgery. An autopsy shows the presence of a pheochromocytoma. In your own words, explain how this incident might have happened.
  2. Your favorite cousin, who is 20 years old, has just been diagnosed with type 2 diabetes. He is overweight and spends most of his time playing computer games or watching television. As a health professional, what advice will you give him?
  3. Your patient has just been diagnosed with SIADH. His mother is asking you what this condition is and what she should expect next. What will you tell her?
  4. Your patient has been admitted to the hospital in preparation for a total removal of her thyroid gland to reverse her hyperthyroidism. She suddenly develops thyroid storm. What manifestations will you find, and what is the pathophysiologic basis of thyroid storm?

Topic Altereations in hormone regulation**

Biology(Hormone)

 
"Looking for a Similar Assignment? Get Expert Help at an Amazing Discount!"

Determining Population Growth Assignment

Determining Population Growth Assignment. In one year, biologists tracking a bear population counted 33 new baby bear cubs in a population of 546 bears.

  1. Calculate the birth rate (b) of this population.
  2. In the same year, the biologists counted 28 black bear deaths. Calculate the death rate for this population.
  3. What is the per capita rate of increase for this black bear population?
  4. What would you estimate the change in black bear population size would be for the following year? Be sure to adjust the N to reflect the new births and deaths. When you get your answer, round to the “nearest bear” and answer the question – is this population increasing or decreasing in size?
  5. Determine the number of births and deaths you would expect the following year. Round to the “nearest bear

Determining Population Growth Assignment

 
"Looking for a Similar Assignment? Get Expert Help at an Amazing Discount!"

: Value Proposition in Patient Care

: Value Proposition in Patient Care. HSA 501 ASSIGNMENT 1

Assignment 1: Value Proposition in Patient Care

Paradise Hospital, Inc. is a for-profit hospital. As the facility’s new hospital administrator, you have been tasked with improving the service value of the hospital. The administration has not done this process since the hospital began operating in the year 1995. The investors are not familiar with the value proposition strategies of hospitals in the current-day America.

Note: You may create and / or make all necessary assumptions needed for the completion of this assignment.

Write a four to six pages paper in which you:

1. Articulate the meaning of value-added service as it pertains to patient care services, and argue the major reasons why it matters to add value to patient services. Justify your response.

2. Outline a system for identifying the functional areas in which changes might be necessary in order to improve the hospital’s service value. Recommend the key methods that you would use to acquire the information necessary to identify the specified functional areas.

3. Specify four (4) specific areas where you believe the administration can add value in Paradise Hospital, and argue the most significant reasons why such value proposition would improve the value of services to the patients.

4. Use five (5) recent (within the last five [5] years) quality academic resources in this assignment. Note: Wikipedia and other websites do not qualify as quality academic resources.

NOTE: PLEASE USE AS A GUIDE OR REWRITE TO MAKE IT YOURS

: Value Proposition in Patient Care

 
"Looking for a Similar Assignment? Get Expert Help at an Amazing Discount!"